P133-S An Improved Protocol of Coupling Synthetic Peptides to KLH for Antibody Production Using DMF

abrf 2007 poster abstracts

JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007 1

Bioinformatics

P1-S

On the Manipulation and Comparison of Protein and Peptide Identification Results from MS Data: Walking on Eggs in the Format JungleP. A. Binz1,2, N. Budin1, A. Niknejad1, A. Masselot1; 1GeneBio, Geneva, Switzerland, 2Swiss Institute of Bioinformatics, Geneva, Switzerland.

A number of protein and peptide identification soft-ware tools based on MS data are available to the proteomics researchers. They all share a common functionality: they process MS data and present in their output peptides and proteins that best match with the input data. Even if restricting to sequence search engines one can observe heterogeneity of approaches, of algorithms, of input param-eters, of the use of available sequence databases, of output information (scores, confidence levels, details of interpreta-tion, etc.) and of possibilities to export results. The results obtained from different tools also vary both from the con-tent and the form point of view. It is a challenge for the bio-informatics to help lab-researchers in manipulating results obtained from replicate analyses or from submissions made to multiple search engines.

Here we present our approach to represent side-by-side results from different MS/MS identification results. We expose elements of the difficulty to get appropriate exports from different search engines and to map the provided information, in order to align it in a single interface. We address questions such as: which export format from each tool is the most useful to perform alignment of results; how to align proteins and peptides coming from two different sequence databases (NCBInr and SwissProt, for instance); how to interpret protein grouping in separate queries; how to identify that proteins are the same if the sequence is not present in the result, or whether any of the database identi-fiers are different, etc.

As an illustrative example, we show how we convert outputs from Phenyx, Mascot, Sequest, or X!Tandem into the Phenyx result comparison feature and more. We will also show how this effort will contribute to and profit from the development of AnalysisXML, a HUPO PSI standard XML format to capture results from protein and peptide identification results.

P2-M

Systematic PTM Analysis of Protein Kinases Using LC-MS/MS DataD. C. Chamrad1, S. Bailey1, A. Wattenberg1, C. Beisenherz-Huss2, R. Graeser2, D. Müller2, M. Blueggel1; 1Protagen AG, Dortmund, Germany, 2ProQinase GmbH, Freiburg, Germany.

Available peptide fragmentation interpretation software is focused on sequence database–driven protein identifica-tion, rather than on primary structure elucidation. Search-ing for posttranslational modification (PTM) or sequence errors currently needs time-critical manual intervention and evaluation. Here we describe the results and perfor-mance of a novel interpretation software, which was used to characterize more than 50 recombinant serine/threonine kinases, receptor tyrosine kinases, or cytoplasmatic tyro-sine kinases.

LC-MS/MS data was acquired after tryptic digestion of the recombinantly produced kinases. The datasets have been imported to the proteome bioinformatics platform ProteinScape. The spectra were screened for a set of modi-fications, amino acid substitutions, unsuspected large mea-surement errors, enzyme no-specificity, and unknown mass shifts. The software restricts the search space by testing only sequences of interest. In widely used sequence data-base searches, testing all modifications and possible non-specific cleavages is not feasable.

Besides the increase in sequence coverage basically caused by detection of one side non-specifically cleaved peptides, numerous modifications were found—namely, phosphorylation. methylation, pyroglutamate formation, methinonine oxidation, and N-terminal acetylation. As spectra of phosphorylated peptides are almost always in the minority compared to their unmodified counterparts, their detection is a challenge, but internal significance analysis revealed a substantial amount of phosphorylation.The phe-nomenon of auto-phosphorylation of kinase proteins was successfully monitored. The phosphorylation sites are cat-egorized according to their sequence motive, and addition-ally their distribution is compared to phosphoryation sites described in public databases. Using this software triggered by the proteome database software proteinscape, searches were performed in a highly automated manner. Manual analysis could be reduced to minutes for the LC-MS/MS datasets containing more than 1000 spectra. Integrated result presentation strategies, which use clustering of spec-tra results on the amino acid level to annotate the protein sequence of interest, avoided the the possibility of seeing excess PTM contained in the large amount of acquired spectra.

poster aBstracts

Key to abstract numbering

Prefixes: P, Poster; RG, Research Group; SP, Scientific Session Presenter; EP, Educational Session Presenter.

Following the hyphen is the designated presentation day:S, Sunday; M, Monday; T, Tuesday.

poster abstracts abrf 2007

2 JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007

P3-T

Quality Control of 2D-Electrophoresis Images Prior to AnalysisA. Borthwick, W. Hudson, M. Lambert, D. Miller, D. Bramwell; Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom.

Management of your image capture process is critical for the success of 2D-electrophoresis gel analysis. Ensuring the consistent quality of scanned images is most problem-atic for large projects involving multiple users at multiple sites. This lack of control will impact on the subsequent detection and quantitation of the images, thus reducing the statistical power of the resulting data analysis.

Most image problems are well known and their causes well understood; for example: incorrect image types, color or compressed images, low spatial resolution, varying bit depth, large and variable image file sizes, low or inconsis-tent use of dynamic ranges, large border variation, uncon-trolled image noise, using the same image more than once, and multiple image orientations.

To help the investigator reduce the occurrence of such problems, we have investigated methods of automatic detection, and propose a standardized way of assessing and rejecting images to avoid subjective decisions that lead to bias. We will show examples of how this process can be used to improve the performance of your capture workflow and thus ensure that you get the best out of your 2D image data.

P4-S

The Mouse Genome Informatics Database: An Integrated Resource for Mouse Genetics and GenomicsC. J. Bult, J. Blake, J. Kadin, J. Eppig, M. Ringwald, J. Richardson, M. Group; The Jackson Laboratory, Bar Harbor, ME, United States.

The Mouse Genome Informatics (MGI; http://www.informatics.jax.org/) database integrates genetic and genomic data with the primary mission of facilitating the use of the mouse as a model system for understanding human biology and disease processes. MGI is the authori-tative source of official mouse genetic nomenclature, gene ontology annotations, mammalian phenotype annotations, and mouse anatomy terms. MGI staff enforce the use of standardized genetic nomenclature, ontologies, and con-trolled vocabularies to describe mouse sequence data, genes, strains, expression data, alleles, and phenotypes. Extensive links between gene-centric information in MGI and other informatics resources (e.g., OMIM, Ensembl, UCSC, NCBI, UniProt) are maintained and updated on a regular basis.

Using the Web-based query interfaces for MGI, users can query for a mouse gene or genes according to diverse biological attributes of those genes, including phenotype associations, gene expression, functional annotation, and genome location. The MGI MouseBLAST server allows users to interrogate the MGI database using nucleotide and/or protein sequences. Functional and phenotypic data from MGI can be viewed in a broader genomic context using

an interactive genome browser called Mouse GBrowse. The power of the MGI database as a research tool for bio-medicine stems from the degree to which data from diverse sources are integrated. Integration, in turn, allows the data to be evaluated in new contexts. For example, integration makes possible such complex queries as “Find all genes from Chromosome 1 where the function is annotated as transcription factor and there is a knockout allele that results in eye dysmorphology.”

The MGI project is supported by NHGRI HG00330, NCI CA89713, and NICHD HD33745.

P5-M

Proteinscape—Software Platform for Managing Proteomics DataD. C. Chamrad1, M. Blueggel1, G. Koerting1, J. Glandorf2, J. Vagts2, P. Hufnagel2, H. Thiele2; 1Protagen AG, Dortmund, Germany, 2Bruker Daltonik GmbH, Bremen, Germany.

Proteomics inherently deals with huge amounts of data. Current mass spectrometers acquire hundreds of thousands of spectra within a single project. Thus, data management and data analysis are a challenge. We have developed a software platform (Proteinscape) that stores all relevant proteomics data efficiently and allows fast access and cor-relation analysis within proteomics projects.

The software is based on a relational database system using Web-based server-client architecture with intra- and Internet access.

Proteinscape stores relevant data from all steps of proteomics projects—study design, sample treatment, separation techniques (e.g., gel electrophoresis or liquid chromatography), protein digestion, mass spectrometry, and protein database search results. Gel spot data can be imported directly from several 2DE-gel image analysis soft-ware packages as well as spot-picking robots. Spectra (MS and MS/MS) are imported automatically during acquisi-tion from MALDI and ESI mass spectrometers.

Many algorithms for automated spectra and search result processing are integrated. PMF spectra are calibrated and filtered for contaminant and polymer peaks (Score-booster). A single non-redundant protein list—containing only proteins that can be distinguished by the MS/MS data—can be generated from MS/MS search results (Pro-teinExtractor). This algorithm can combine data from dif-ferent search algorithms or different experiments (MALDI/ESI, or acquisition repetitions) into a single protein list.

Navigation within the database is possible either by using the hierarchy of project, sample, protein/peptide sep-aration, spectrum, and identification results, or by using a gel viewer plug-in. Available features include zooming, annotations (protein, spot name, etc.), export of the anno-tated image, and links to spot, spectrum, and protein data.

Proteinscape includes sophisticated query tools that allow data retrieval for typical questions in proteome proj-ects. Here we present the benefit and power of usage of 6 years of continuous use of the software in over 70 proteome projects managed in house.


JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007 �

P6-T

Tranche: Secure Decentralized Data Storage for the Proteomics CommunityJ. A. Falkner, P. C. Andrews; University of Michigan, Ann Arbor, MI, United States.

The number, size, and format variation for proteomics data files (both raw and processed), annotation, as well as challenges in designing a robust data repository are some of the major factors inhibiting public dissemination of pro-teomics data. Sharing large amounts of data and software is a legitimate need in the field of proteomics and other scientific disciplines as replication of results and the ben-efits of data reanalysis relies heavily on having access to the original data. Several journals have already published rec-ommendations for providing access to data associated with proteomics manuscripts; however, researchers have been left with the challenge of how to appropriately satisfy the recommendations. Of particular concern is how potentially large datasets (gigabytes to terabytes of raw data) may be efficiently hosted in a publicly accessible fashion.

Described here is Tranche, a secured peer-to-peer sys-tem (http://www.proteomecommons.org/dev/dfs/), along with a reference implementation, supported by Proteome-Commons.org, that is capable of hosting virtually unlim-ited amounts of data and supporting virtually unlimited users. Furthermore Tranche solves many of the prominent concerns in data dissemination, including hosting raw data associated with a proteomics experiment and maintaining annotation. It is intended as both a reference implemen-tation and a model system for comparison to other pro-teomics data dissemination efforts. Tranche currently hosts many prominent proteomics datasets and mirrors of other proteomics data resources, including most all of the pub-licly available proteomics data.

P7-S

Combining Workflow-Based Project Organization with Protein-Dependant Data Retrieval for the Retrieval of Extensive Proteome InformationJ. Glandorf1, H. Thiele1, M. Macht1, O. Vorm2, A. Podtelejnikov2; 1Bruker Daltonik GmbH, Bremen, Germany, 2Proxeon Biosystems A/S, Odense, Denmark.

In the course of a full-scale proteomics experiment, the handling of the data as well as the retrieval of the relevant information from the results is a major challenge due to the massive amount of generated data (gel images, chromato-grams, and spectra) as well as associated result information (sequences, literature, etc.). To obtain meaningful infor-mation from these data, one has to filter the results in an easy way. Possibilities to do so can be based on GO terms or structural features such as transmembrane domains, involvement in certain pathways, etc.

In this presentation we will show how a combination of a software package with a workflow-based result orga-nization (Bruker ProteinScape) and a protein-centered data-mining software (Proxeon ProteinCenter) can assist in the comparison of the results from large projects, such as comparison of cross-platform results from 2D PAGE/MS with shotgun LC-ESI-MS/MS. We will present differences

between different technologies and show how these differ-ences can be easily identified and how they allow us to draw conclusions on the involved technologies.

P8-M

Bioinformatic Analysis of Neural Stem Cell DifferentiationL. A. Goff1, R. Hart1, R. Jornsten1, S. Keles2; 1Rutgers University, Piscataway, NJ, United States, 2University of Wisconsin, Madison, WI, United States.

We analyzed mRNAs regulated during differentiation of rat neural stem cells using the ABI1700 microarray plat-form. This microarray, while technically advanced, suffers from the difficulty of integrating hybridization results into public databases for systems-level analysis. This is particu-larly true for the rat array, since many of the probes were designed for transcripts based on predicted human and mouse homologs. Using several strategies, we increased the public annotation of the 27,531 probes from 43% to over 65%. To increase the dynamic range of annotation, probes were mapped to numerous public keys from several data sources. Consensus annotation from multiple sources was determined for well-scoring alignments, and a confi-dence-based ranking system established for probes with less agreement across multiple data sources. Previous attempts at genomic interpretation using the Celera annotation model resulted in poor overlap with expected genomic sequences. Since the public keys are more precisely mapped to the genome, we could now analyze the relationships between predicted transcription factor binding sites and expression clusters. Results collected from a differentiation time course of two neural stem cell clones were clustered using a model-based algorithm. Transcription factor bind-ing sites were predicted from upstream regions of mapped transcripts using position-weight matrices from either JAS-PAR or TRANSFAC, and the resulting scores were used to discriminate between observed expression clusters. A classification and regression tree analysis was conducted using cluster numbers as gene identifiers and TFBS scores as predictors, pruning back to obtain a tree with the low-est gene class prediction error rate. Results identify several transcription factors, the presence or absence of which are sufficient to differentiate clusters of mRNAs changing over time from those that are static, as well as clusters describing cell-line differences. Public annotation of the ABI1700 rat genome array will be valuable for integrating results into future systems-level analyses.


� JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007

P9-T

ProteinExtractor—From Peptide ID to Protein IDG. Koerting1, C. Stephan2, K. Marcus2, D. C. Chamrad1, P. Hufnagel3, U. Schweiger-Hufnagel3, J. Glandorf3, H. E. Meyer2, H. Thiele3, M. Blueggel1; 1Protagen AG, Dortmund, Germany, 2Medizinisches Proteom Center, Bochum, Germany, 3Bruker Daltonik GmbH, Bremen, Germany.

In proteomics workflows, proteins are often digested first, then peptides are separated and subjected to iden-tification by mass spectrometry (e.g., 2D-LC). In this process the peptide assignment to a protein is lost and has to be rebuilt by bioinformatic methods. We present ProteinExtractor, a module of the ProteinScape Bioinfor-matics Platform, which uses an empiric, iterative method to derive minimal protein lists from peptide search results, which may even come from different search algorithms or different MS datasets.

ProteinExtractor uses an iterative approach to generate a minimal protein list. With composite database searches ProteinExtractor allows measuring the false-positive rate of the protein list. A test dataset (five recombinant pro-teins, 408 spectra, Bruker Ultraflex), and a real-life dataset (200410 LC/ESI-MS/MS spectra, Bruker Esquire HCT-Ultra, and 11619 LC/MALDI-MS/MS spectra, Bruker Ultraflex, both obtained from an analysis of proteins from a human cell line—SW480) were analyzed.

The most probable protein sequence entries contained in the test dataset were identified with intensive manual data interpretation by several mass spectrometry experts. Using standard search algorithms, the correct protein sequence database entries are scattered over the first 171 protein ranks. Together with application specialists, we developed a set of rules to define a minimal protein list containing only those proteins (and isoforms) that can be unequivocally distinguished on the basis of MS/MS data. Applying these rules, the correct five proteins are ranked within the top eight protein candidates.

In the real-life dataset, the peptide search results of Mascot, Sequest, Phenyx, and ProteinSolver were merged using ProteinExtractor. Merging all four search algorithms, over 50% more proteins could be identified than by using Mascot alone (with a false-positive rate of less then 2.5%). Merging ESI and MALDI data together, another 25% more proteins could be identified.

P10-S

A Novel Protein Database Search Algorithm for 1D and 2D LC-MS DataG. Li, D. Golick, B. Dyson, J. C. Silva, H. Liu, J. P. C. Vissers, J. C. Gebler, M. V. Gorenstein, S. J. Geromanos; Waters Corporation, Milford, MA, United States.

A novel, “Ion Accounting” algorithm has been devel-oped for protein identification using time-resolved, LC-MSE data from 1D and 2D LC-MS experiments. The data from a 1D LC-MS analysis generate a series of precursor-product tables that are initially queried against a protein database using the “Ion Accounting” algorithm. Hereby each precursor and product is associated with only single

peptide identification. The database search is a hierarchal process containing three modules. With the first module, the data are matched to only correctly cleaved proteolytic peptides whose precursor and product ion mass tolerances are within 10 and 20 ppm, respectively. With the second module, precursor and product ions that have not yet been assigned are queried against a subset database of the identi-fied proteins from the first module. The second module includes missed cleavages, in-source fragments, neutral losses, and variable modifications. With the last module, the remaining unidentified ions are considered against the complete database for additional protein identifications (including PMF) with improved selectivity and specificity from the elimination of those precursor and product ions from the first two modules.

The data from a 2D LC-MS separation of proteolytic peptides is conducted by fractionating the peptides in a first dimension and subsequent separation in a second dimen-sion during the LC-MSE analysis. Each fraction produces a series of precursor-product tables. From these tables, the peptides (precursor-products) that were not distributed over multiple fractions are saved to a “Combined Precursor-Product Table” (CPPT). The peptides that are split among neighboring fractions are combined by precursor mass, precursor retention time, and product ion pattern, and are appended to the CCPT. The final CCPT is submitted to the “Ion Accounting” protein database search engine in a similar fashion to the 1D LC-MS data analysis.

P11-M

Comprehensive and Reliable Proteome Analysis Using Bioinformatic Strategies for Automated Result ValidationM. Macht1, C. Albers1, K. Sparbier2, A. Asperger1, J. Glandorf1, H. Thiele1; 1Bruker Daltonik GmbH, Bremen, Germany, 2Bruker Daltonik GmbH, Leipzig, Germany.

Proteomic analyses typically produce massive amounts of mass spectrometric data, which are analyzed in an auto-mated way by database search engines for retrieval of pep-tide sequences and subsequent inference on the correspond-ing protein sequences. However, this process turned out to be error prone, producing false positives and multiple hits for the same proteins for various reasons.

In this study we analyzed the human serum glyco-subproteome. For this, glycoproteins were produced in five separate extractions by affinity interaction chromatogra-phy using magnetic beads coated with the lectins ConA, WGA, LCA, and AIA, as well as with boronic acid. The eluates from the beads were digested using trypsin and sub-sequently analyzed by LC-MALDI-MS/MS as well as LC-ESI-MS/MS. The analyses were carried out as up to triple replicates. All the data were submitted to the ProteinScape database system.

Within ProteinScape, the datasets were searched against Mascot and Phenyx. The resulting peptide identifications were analyzed by the ProteinExtractor tool to reduce the list by the false positives using a decoy strategy, and sub-sequently merged into a combined list of identified pro-teins for the respective sample preparations. This allows the combination of data from different search engines



as well as from different experiments (ESI and MALDI). The use of decoy strategies as well as application of the ProteinExtractor to overcome the protein inference prob-lem minimizes the need for manual validation (which is nevertheless easily possible using raw spectra information). In parallel, the use of a single data repository allows for easy access to the combined information from different work-flows and links to external tools complement the system for project-spanning comparisons of datasets.

P12-T

Prediction of Proteotypic Peptide Candidates for MRM AnalysisK. Marcus1, R. Reinhardt2, E. Langenfeld1, H. E. Meyer1, M. Blüggel2; 1Medizinisches Proteom Center, Bochum, Germany, 2Protagen AG, Dortmund, Germany.

The application of multireaction monitoring (MRM) for proteomics analysis is a quite recent development. The sen-sitivity of four orders of magnitude, reproducibility, and the option of quantification as well as high throughput make MRM a valuable tool for measuring specific proteins.

We present a bioinformatics workflow for the deter-mination of MRM candidates that reduces the number of nonproteotypic peptide candidates considerably, enabling the analysis of complex and highly homologous protein families.

A peptide candidate has to be unique for its targeted protein in respect to the proteome of the organism. Poly-morphisms of the target protein are an important issue, when coverage of all alleles of the target protein is desired rather than rudimentary genotyping by MS. Both aspects can be solved by using annotated protein databases. When using absolute quantification (e.g., the AQUA technique of Kirkpatrick et al.), the ability of synthesis for the sta-ble isotope-labeled peptides has to be taken into account additionally. The selection of suitable fragment ions can be done by evaluating previously acquired spectra or MS/MS fragment prediction.

The result has been compared to measurements of human cytochrome P450 (CYP450). CYP450 comprises families (over 200 protein members known today) with low evolutionary conservation and thus high homology. Devel-oping antibodies specific for one protein can be a daunt-ing task; this holds especially true for the human CYP2 family. By targeting only proteotypic peptides using mass spectrometry, antibody-related problems are avoided. As a proof of concept, the analysis of highly polymorphic human CPY2D6 is depicted by ESI-MRM/MS/MS analysis.

P13-S

Database Protein Information Searching Engine via Internet: PIKEJ. Medina-Aunon1, M. Macht2, A. Quinn3, J. Albar1, J. Glandorf2, H. Thiele2; 1ProteoRed, CNB-CSIC, Madrid, Spain, 2Bruker Daltonik GmbH, Bremen, Germany, 3European Bioinformatics Institute, Hinxton, United Kingdom.

One of the main goals in proteomics is to extract and collect all the functional information available in existing databases in relation to a defined set of identified proteins. Due to the huge amount of data available, it is not possible to gather up this information by hand; we need to have automatic methods for addressing this task.

Protein information and knowledge extractor (PIKE) solves this problem by accessing several public information systems and databases automatically through the Internet and retrieving all functional information available on the different repositories, and then clustering this information according to the pre-selected criteria. The PIKE bioin-formatics tool, accessible through http://proteo.cnb.uam.es:8080/pike, uses the Java and XML languages. Starting with a selected group of identified proteins, listed as NCBI nr, uniprot, and/or ipi (http://www.ebi.ac.uk/IPI/IPIhelp.html) accession codes, PIKE retrieves all relevant informa-tion stored in databases by choosing the correct pathway and/or the best information source.

Once the search is done, a typical PIKE output shows a report table with an entry for each protein containing all extracted information. The report contains a large amount of meaningful protein features, such as (1) function infor-mation, (2) sub-cellular location, (3) tissue specificity, (4) links with other repositories, such as Mendelian Inheri-tance in Man (OMIM) or Kyoto Encyclopaedia of Genes and Genomes (KEGG), and (5) gene ontology tree classifi-cation. The table is exportable in CSV and text file formats, and, more important, it is possible to export it in PRIDE XML (http://www.ebi.ac.uk/pride/) format for results inte-gration into the information stored in other applications such as ProteinScape.

P14-M

PRIME: Proteome Research Information Management Environment For High-Throughput Proteomics LaboratoriesP. G. Papoulias, D. Lentz, P. C. Andrews; University of Michigan, Ann Arbor, MI, United States.

Proteomics laboratories and proteomics service facilities produce a large volume of data that originate from diverse sources, involve the participation of several members of the lab, and are communicated to clients and collaborators of the laboratory. Laboratory information management sys-tems (LIMS) play a key role in collating and tracking the flow of data through the laboratory. The cost of off-the-shelf LIMS systems can be significant and compounded by further customization, development, and installation costs. Fields that employ rapidly changing technologies in sup-port of research require information management that is stringent and secure, but also must be adaptable.


� JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007

We have developed an open-source scientific data man-agement system for proteomics that is capable of acting as a stand-alone LIMS system, or working in conjunction with other LIMS systems. We support sample processing workflow, protocols, and datasets generated by LC-ESI, LC-MALDI, MALDI, 1D-GELS, 2D-GELS, and 2D-gel image analysis. The workflow supports input from existing data analysis and allows batch analysis of proteomics data to be queued to search engines and other tools. It supports extensive data curation, has a simple Web browser interface that allows tiered, secure access to data and functions, and also allows export of data from individual spectra up to full project files. The source code is available from Proteo-mecommons.org. Installation of the software can be per-formed through https://www.prime-sdms.org/main.htm.

P15-T

SameSpots: Validating a Novel Approach to 2D Electrophoresis Image AnalysisI. Reah, M. O’Gorman, A. Borthwick, D. Miller, D. Bramwell; Nonlinear Dynamics Ltd, Newcastle upon Tyne, United Kingdom.

2D-PAGE experiments can provide a powerful means of investigating protein expression behavior in a cell or tis-sue across different disease states or other experimental conditions. Traditional analysis of 2D experiments typi-cally requires a large amount of post-detection editing in order to prepare the data for statistical research. With the application of SameSpots, all protein spots are fully matched and thus no missing values exist. This enables a more robust statistical exploration of the data with a reduc-tion in subjective editing.

The SameSpots workflow has a semi-automatic gel alignment step and uses the arcsinh transform introduced by Huber et al.1 for inter-gel calibration and variance stabili-zation (VSN). To validate these techniques we repeated the work of Nishihara and Champion2 and Karp and Lilley.3 The results of this study demonstrate that gel alignment has no adverse affect on spot volume quantitation, and we also show that VSN outperforms traditional normalization and variance stabilizing methods used in 2D gel analysis.

references

1. Huber W, Von Heydebreck A, Sültmann H, Poustka A, Vin-gron M. Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics 2002;18 suppl. 1:S96–S104

2. Nishihara JC, Champion KM. Quantitative evaluation of pro-teins in one- and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis 2002;23:2203–2215

3. Karp NA, Lilley KS. Maximising sensitivity for detecting changes in protein expression: Experimental design using Mini-mal CyDyes. Proteomics 2005;5 (12):3105.

P16-S

Database Search of High Mass Resolution DataR. G. Sadygov, V. Zabrouskov; ThermoFisher Scientific, San Jose, CA, United States.

The work describes modifications to and application of Sequest database search algorithms to identify peptides from their high mass accuracy tandem mass spectra. We show the technical problems encountered when attempting to use high mass accuracy data with the original Sequest algorithm. To overcome the problems, modifications have been made to the algorithm. The modifications are such that the results from the unmodified and modified algo-rithms are the same for the unit mass accuracy data. The work presents advantages in terms of the speed and reduced memory requirements of the modified algorithm.

We apply the modified algorithm to a dataset obtained from site-specific digestion of a set of known proteins. The results demonstrate the effect of high mass accuracy on such characteristics of the Sequest scoring as cross-correla-tion score and delta-Cn. Based on the correlations between the mass accuracy and the Sequest scores, we attempt to identify optimal conditions for the mass accuracy to be applied in the database searches. A comparative analysis with the dataset of the same proteins, but under unit mass accuracy conditions, will also be presented. We analyze results obtained from searches of the spectra against the reversed databases under the high mass accuracy condi-tions.

P17-M

Improving Sensitivity by Combining Results from Multiple MS/MS Search Methodologies with the Scaffold Computer AlgorithmB. C. Searle, M. Turner; Proteome Software, Inc., Portland, OR, United States.

Database-searching programs generally identify only a fraction of the spectra acquired in a standard LC/MS/MS study of digested proteins. Subtle variations in database-searching algorithms of MS/MS spectra have been known to provide different identification results. To leverage this variation, we developed Scaffold to probabilistically combine the results of multiple search engines, including



Sequest, Mascot, and X!Tandem. Here we present a “tell all” explanation of the specific methodology behind Scaffold that converts scores into search engine independent peptide probabilities. These probabilities can be readily combined across search engines using Bayesian rules and the Expec-tation Maximization learning algorithm. We demonstrate how we normally gain 20% to 100% more highly confident (>95%) MS/MS spectrum identifications with each addi-tional search engine, which is primarily due to increased confidence in low-scoring matches. We also show that this method works reliably across a variety of search engines and instrumentation platforms without re-tuning.

P18-T

MassSieve: A New Tool for Mass Spectrometry–Based ProteomicsD. J. Slotta, M. McFarland, A. Makusky, S. Markey; NIMH/NIH, Bethesda, MD, United States.

The success of peptide sequence assignment algorithms such as OMSSA and Mascot for mass spectrometry has led to the need for a tool to evaluate the results. DBParser is such a software tool, previously developed by the Labora-tory of Neurotoxicology (LNT) lab for this purpose. Its value for parsimonious analysis of proteins associated with experiments has led to its use for analyzing larger datasets than initially anticipated (hundreds of data files with mil-lions of spectra). MassSieve builds on this experience and is designed as open-source protein assignment software that can be scaled to apply parsimony principles to very large experiments without dataset size limitations. In addition it allows a more interactive view of the results.

P19-S

Managing Proteomics Data from Data Generation and Data Warehousing to Central Data Repository and Journal Reviewing ProcessesH. Thiele1, J. Glandorf1, G. Koerting2, K. Reidegeld3, M. Blüggel2, H. Meyer3, C. Stephan3; 1bruker Daltonik GmbH, Bremen, Germany, 2Protagen AG, Dortmund, Germany, 3Medical Proteom-Center, Ruhr Universität Bochum, Germany.

In today’s proteomics research, various techniques and instrumentation bioinformatics tools are necessary to man-age the large amount of heterogeneous data with an auto-matic quality control to produce reliable and comparable results. Therefore a data-processing pipeline is manda-tory for data validation and comparison in a data-ware-housing system. The proteome bioinformatics platform ProteinScape has been proven to cover these needs. The reprocessing of HUPO BPP participants’ MS data was done within ProteinScape. The reprocessed information was transferred into the global data repository PRIDE.

ProteinScape as a data-warehousing system covers two main aspects: archiving relevant data of the proteomics workflow and information extraction functionality (protein identification, quantification and generation of biological knowledge). As a strategy for automatic data validation, dif-

ferent protein search engines are integrated. Result analysis is performed using a decoy database search strategy, which allows the measurement of the false-positive identification rate. Peptide identifications across different workflows, different MS techniques, and different search engines are merged to obtain a quality-controlled protein list.

The proteomics identifications database (PRIDE), as a public data repository, is an archiving system where data are finally stored and no longer changed by further process-ing steps. Data submission to PRIDE is open to proteomics laboratories generating protein and peptide identifications. An export tool has been developed for transferring all rel-evant HUPO BPP data from ProteinScape into PRIDE using the PRIDE.xml format.

The EU-funded ProDac project will coordinate the development of software tools covering international stan-dards for the representation of proteomics data. The imple-mentation of data submission pipelines and systematic data collection in public standards–compliant repositories will cover all aspects, from the generation of MS data in each laboratory to the conversion of all the annotating infor-mation and identifications to a standardized format. Such datasets can be used in the course of publishing in scien-tific journals.

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NCI CGEMS Data Portal: Sharing Data for Genome-Wide Association StudiesL. Yang; National Cancer Institute, Rockville, MD, United States.

A new NCI initiative, Cancer Genetic Markers of Sus-ceptibility (CGEMS), is a three-year study designed to identify common genetic variations associated with risk for prostate and breast cancer. CGEMS will analyze the entire genome for the most common type of genetic variation, the single-nucleotide polymorphism (SNP). By studying large populations of individuals with and without disease, the CGEMS research can provide powerful indicators as to which SNP variations are associated with each disease. This study design is especially valuable for unraveling the genetic origins of complex diseases such as prostate and breast cancer.

A critical requirement of the CGEMS project is to share raw data and analysis results from the study with the cancer research community. The NCI Center for Bioinformatics (NCICB), in collaboration with other NCI research groups, has built the CGEMS data portal to support data shar-ing of the CGEMS project (https://caintegrator.nci.nih.gov/cgems/).

The first whole genome scan includes approximately 1200 prostate cancer cases and 1200 controls. The datasets available through the portal include:

• Association test results for over 300,000 SNPs • Frequency and descriptive statistics on these 300,000

SNPs • Individual phenotypic and genotypic data for the study

participants and control samples. Note that these data can be made available only to eligible investigators after a registration process.



CGEMS data portal development has leveraged the caIntegrator application framework, developed at NCICB. It shares a common set of application programming inter-faces (APIs) and specification objects that support the clini-cal genomic analysis services. This allows fast development of Web-based query functionalities on all the data objects from the CGEMS project.

BiomarKers

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The Application of Multivariate Model Building to Derive Predictive ‘Signatures’ from Proteomics DataD. Bramwell1, I. Morns1, M. O’Gorman1, S. Hoving2, B. Wiedmann2, H. Voshol2; 1Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom, 2Novartis Institutes for BioMedical Research, Basel, Switzerland.

Objective: To apply advanced statistical model-building procedures to derive proteomic “signatures” from 2D gels and validate the approach by predicting double-blinded samples.

Methods: A large experiment was used to explore the power of the predictive modeling process (340 samples, 18 groups, seven double-blinded and three unknown).

The images were geometrically corrected and then analyzed at a pixel level. On completion of this procedure, areas important for obtaining good group discrimination were automatically identified.

The areas were ranked and visually examined. Up to 10 per group were selected for the next stage of analysis. The 117 resultant spots were then used to build predictive models. The models were explored in the context of the experiment and also for their prediction performance. This process enables the selection of candidate spots that may be below standard univariate thresholds (such as p < 0.05, 1.5-fold change).

Results: Models were successfully built that gave perfect performance on the training sets. The blind samples were successfully predicted and interesting information on the unknown samples was produced and is the subject of fur-ther experimentation. The effective “systems” dimension for the 18 group sets was estimated to be 12, which suggests we may have more groups than is supported by the data. A “minimal spot set” was calculated and showed a saturation in prediction performance at around 60 spots. A follow-on procedure was employed to choose the best spots for group discrimination and also to specify the spot number vs. per-formance relationship.

Conclusion: Proteomics data provide a rich source for advanced statistical modeling techniques, and using stan-dard double-blind procedures can add an intuitive confi-dence to the experimental results. The techniques are very powerful in assisting in the exploration of the complex rela-tionships intrinsic to the data.

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LC-MALDI Top-Down Biomarker Profiling and IdentificationS. Brand, M. Meyer, S. Hahner, D. Suckau; Bruker Daltonik GmbH, Bremen, Germany.

The search for new and validated biomarkers is of par-ticular interest in clinical areas such as oncology, neurology, toxicology, and pharmacology. One of the challenges in finding the right technology for biomarker research is to combine a statistically reasonable throughput—hundreds of samples like serum, plasma, cell lysate, and urine—with an in-depth proteome technology. As proteolytic events play a significant role, in particular in disease-related events, biomarker discovery approaches may benefit from a top-down profiling approach, as proteolytic isoforms remain intact during the analysis.

We present the combination of sample preparation based on magnetic nanoparticles purification (wax) or other pre-separation methods with the high-resolution HPLC-MALDI analysis of the undigested peptides and proteins. Proof-of-principle experiments included 36 sam-ples in three groups spiked with different concentrations of a marker peptide. Multivariate statistics (PCA) achieved a proper grouping of the samples and detected the spiked material correctly from the complex matrix. Subsequent MS/MS spectra allowed the identification.

First experiments clearly demonstrate that this technol-ogy significantly increases the number of detectable signals received from human serum (>1500) and is very reproduc-ible. Therefore, this approach opens the door for high-throughput, in-depth analysis of clinical samples for the detection of biomarkers. Furthermore the marker detec-tion does not require previous identification (i.e., uncom-mon structures can qualify to be markers) and the overall MS/MS workload is extremely reduced. Finally the reduc-tion of protein complexity per fraction after LC-MALDI separation allows the use of a simple and fast method for the identification of biomarker candidates: in situ digestion provides for protein identification directly from LC runs collected on MALDI targets. Thus we narrowed the gap between detection of biomarker candidates and their final identification. This identification is mandatory for valida-tion and for any further diagnostic use of biomarkers.

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Limit of Quantitation for Low-Abundance Proteins in Plasma by Targeted MSM. Burgess, H. Keshishian, E. Kuhn, T. Addona, S. A. Carr; Broad Institute, Cambridge, MA, United States.

Biomarker discovery results in the creation of candi-date lists of potential markers that must be subsequently verified in plasma.1 The most mature methods at present require abundant protein depletion and fractionation at the protein/peptide levels in order to detect and quantitate low ng/mL concentrations of plasma proteins by stable isotope dilution mass spectrometry. Sample-processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate bio-marker proteins were evaluated by adding five non-native



proteins to immunoaffinity-depleted female plasma at varying concentrations (1000, 100, 50, 25, and 10 ng/mL). Each protein was monitored by one or more representa-tive synthetic tryptic peptides labeled with [13C6]leucine or [13C5] valine. Following reduction, carbamidomethylation, and enzymatic digestion, two separate processing paths were compared. In path 1, digested plasma was diluted 1:10 and [13C] internal standards were added just prior to direct analysis by multiple reaction monitoring with LC-MS/MS (MRM LC-MS/MS). In path 2, peptides were separated by strong cation exchange, and [13C] internal standards were added to corresponding SCX fractions prior to analy-sis by MRM LC-MS/MS. Detection and quantitation by MRM used the response of at least two product ions from each of the signature peptides. Using processing path 1, we achieved detection and quantitation down to 50 ng/mL in depleted plasma. However, using processing path 2, we achieved detection and quantitation of all spiked proteins, including the non-native protein at 10 ng/mL. While anal-ysis of non-fractionated plasma achieved higher recovery of those proteins detected in both processes, SCX fraction-ation at the peptide level clearly increases detection and LOQs for potential biomarker proteins in plasma.

reference

1. Rifai N, Gillette MA, Carr SA. Protein biomarker discovery and validation: The long and uncertain path to clinical utility. Nat Biotechnol 2006;24:971–983.

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Quantitative Proteomics of Formalin-Fixed Archival TissueM. Darfler1, B. Hood2, T. Guiel1, T. Conrads3, T. Veenstra3, D. Krizman1; 1Expression Pathology Inc., Gaithersburg, MD, United States, 2SAIC-Frederick, Inc., Frederick, MD, United States, 3SAIC-Frederick, Frederick, MD, United States.

Capabilities for quantitative mass spectrometry–based proteomic profiling of formalin-fixed archival tissue have been developed. Using Liquid Tissue reagents and protocols, we have effectively profiled and validated known and novel cancer biomarkers across a wide variety of fixed cancer tis-sue samples. Recently developed applications include quan-tification of protein expression in fixed archival tissue with an emphasis on oral cavity (head and neck squamous cell carcinoma—HNSCC) and breast cancer. Spectral count bioinformatics provides a non-labeling method for quanti-tative proteomics of formalin-fixed histologically-defined oral cavity cancer. Results indicate many proteins were differentially expressed in cells obtained by laser microdis-section of normal, highly differentiated, moderately dif-ferentiated, and poorly differentiated HNSCC fixed tissue. Candidate protein biomarkers found to be differentially expressed in the process of HNSCC progression were con-firmed and validated by immunohistochemistry on large panels of HNSCC tissue. In addition, we have developed and evaluated a method for direct detection and absolute quantification by selected reaction monitoring (SRM) of HER2 directly in formalin-fixed paraffin-embedded

breast cancer tissue using a stable isotope standard peptide derived from HER2. Soluble protein extracts from a col-lection of breast cancer tissues known to express a range of HER2 were prepared using Liquid Tissue reagents, and quantitative levels were determined. Results demonstrate the ability to quantitate HER2 expression in Liquid Tis-sue extracts from fixed tissue sections that correlate with standard IHC and indicate the ability to quantify HER2 in immunohistochemical-negative cells. These cumulative results demonstrate development of technologies for quan-titative proteomic analysis of proteins that can be applied to the vast worldwide formalin-fixed tissue archives.

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Gene Expression Profiling from Formalin-Fixed, Paraffin-Embedded Tissues Using the QuantiGene Branched DNA AssayJ. Davies, B. Maqsodi, W. Yang, Y. Ma, Y. Luo, G. McMaster; Panomics, Inc., Fremont, CA, United States.

Large numbers of formalin-fixed, paraffin-embedded (FFPE) human tissue specimens with known clinical out-come are archived worldwide, representing a vast resource for biomarker and gene-disease association studies. How-ever, RNA quality in FFPE tissues is compromised by chemical modifications and extensive fragmentation caused by formalin fixation. As a result, quantifying RNA in FFPE samples can be problematic.

Here we describe application of the QuantiGene branched DNA (bDNA) assay to gene expression profiling of FFPE samples. This hybridization-based, signal ampli-fication method measures RNA directly from FFPE tis-sue homogenates, avoiding variability introduced by RNA extraction as well as biases inherent to reverse transcription and amplification of target sequences.

We show that the QuantiGene assay is insensitive to chemical modifications introduced by formalin fixation and can efficiently capture even highly degraded RNA. Small changes in gene expression are reliably measured even in FFPE tissues stored for more than 10 y. Results from a large number of independent studies, in which the QuantiGene assay was used to quantify more than 220 RNA targets in nearly 400 FFPE samples from nine dif-ferent tissues, will be summarized. Data comparing the accuracy, precision, and sensitivity of QuantiGene and real-time, quantitative PCR (RT-qPCR) assays using FFPE samples will be presented.

Additionally, we discuss the usefulness of parallel QuantiGene assays for assessment of FFPE tissue homog-enate quantity and RNA quality, as well as improved meth-ods for normalization of data from FFPE samples.

Finally, we demonstrate the feasibility of simultaneous, multiplexed RNA measurements from FFPE tissue homog-enates using the QuantiGene Plex Reagent System.

Thus the QuantiGene FFPE Reagent System is a com-prehensive solution for gene expression analysis of FFPE tissues, allowing retrospective studies that were previously not feasible.



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Detection of Candidate Protein Biomarkers in Human Serum by Multiple Reaction Monitoring: Improved Limits of Detection and QuantificationC. Doneanu, W. Chen, A. Chakraborty, J. Gebler; Waters Corporation, Milford, MA, United States.

Mass spectrometry–based biomarker discovery in bio-fluids produces a list of candidate proteins that must be verified and quantified in a large number of samples before a candidate becomes a useful diagnostic, prognostic, or pharmacodynamic marker. Because of the high sensitiv-ity and specificity provided by multiple reaction monitor-ing (MRM), this MS/MS method has recently been used for verification and quantification of potential biomakers. However, the wide dynamic range of protein concentra-tions in serum prohibits direct detection of many useful biomarkers at the concentration level of low nanogram/mL to picogram/mL range without any sample fractionation and/or enrichment.

In this presentation, we evaluate the utility of two sam-ple enrichment techniques for improving the limit of detec-tion and limit of quantification (LOQ) for MRM analysis of several candidate protein biomarkers.

In the first sample enrichment method, we used immuno-depletion to remove either the six most abundant serum proteins (90% serum depletion) or the twenty most abundant proteins (97% serum depletion) before MRM analysis of low-abundance potential biomarkers. The sec-ond sample enrichment method that we evaluated was the glycoprotein affinity enrichment method. Several low-abundance serum proteins were quantified by the MRM method using a triple quadrupole mass spectrometer cou-pled to a nanoscale liquid chromatograph. The effects of immuno-depletion and affinity enrichment on the LOQ of selected candidate proteins biomarkers in human plasma were compared.

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Evaluation of Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl Ether (‘Compound A’) Effects on Urine Protein Excretion in Rats Using Mass SpectrometryK. Dong1, M. S. Minkoff1, J. D. Miller1, E. D. Kharasch2; 1Applied Biosystems, Framingham, MA, United States, 2Department of Anesthesiology, Washington University, St. Louis, MO, United States.

Fluoromethyl-2,2-dif luoro-1-(trif luoromethyl)vinyl ether (FDVE or “compound A”), a haloalkene degradant of the volatile anesthetic sevoflurane, is nephrotoxic in rats. FDVE bioactivation mediates the toxicity, but the molecu-lar and cellular mechanisms of toxification are unknown. FDVE caused rapid and brisk changes in kidney gene expression, providing potential insights into mechanisms of toxicity, and potential biomarkers for nephrotoxicity.1 Nev-ertheless, it is unknown whether gene-expression changes are reflected in protein expression, or whether such tissue changes would be reflected in excreted urine proteins. This investigation was to evaluate FDVE effects on urine protein excretion using mass spectrometry.

After Animal Use Committee approval, male Fisher 344 rats (250–300 g) housed in individual metabolic cages received a single intraperitoneal injection of 0.25 mmol/kg FDVE, and all urine was collected daily for 1 wk, as described previously.2

The samples were labeled with iTRAQ reagents, and both the 4800 MALDI TOF/TOF Analyzer and the 4000 Q TRAP system (AB/MDS SCIEX) were used to acquire data in MS and MS/MS modes. Data were processed with MarkerView software and ProteinPilot Software (AB/MDS SCIEX).

The results demonstrate that FDVE causes certain alter-ations in urine protein/peptide excretion. Multiple com-ponents were differentially expressed in a time-dependent manner. Excretion of several endogenously excreted pro-teins was rapidly decreased by FDVE. Other native pep-tides showed increased excretion following FDVE, and then gradually decreased to pre-dose levels. Excretion of a third set of proteins/peptides, minimally or not detect-able in controls, was upregulated following FDVE. Further experiments will be conducted to identify the protein/pep-tide markers using LC MALDI MS/MS and other tech-nologies to further investigate the usefulness of MS for identifying biomarkers for FDVE nephrotoxicity.

Supported by NIH DK53765

references

1. Kharasch ED. Gene expression profiling of nephrotoxicity from the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (“Compound A”) in rats. Toxicol Sci 2006;90:419–31

2. Sheffels P, Schroeder JL, Altuntas TG, Liggitt HD, Kharasch ED. Role of cytochrome P4503A in cysteine S-conjugates sulf-oxidation and the nephrotoxicity of the sevoflurane degradation product f luoromethyl-2,2-dif luoro-1-(trif luoromethyl)vinyl ether (“compound A”) in rats. Chem Res Toxicol 2004;17:1177–89.

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Digging Deeper and Faster into Proteome by IgY-Immunoaffinity FractionationX. Fang1, L. Huang1, S. Sikora1, D. Hinerfeld2, S. Tam2, P. Gagné3, G. G. Poirier3, C. Kusumoto 4, K. Obata4, D. Q. Yang5, W. Zhang1; 1GenWay Biotech, Inc., San Diego, CA, United States, 2University of Massachusetts Medical School, Shrewsbury, MA, United States, 3Laval University, Québec City, PQ, Canada, 4PSS Bio Instruments, Inc., Livermore, CA, United States, 5PSS Bio Instruments, Inc., Gaithersburg, MD, United States.

After separating the highly abundant proteins (HAP) by IgY affinity column, the next layer of abundant pro-tein, moderately abundant proteins (MAP), becomes an obstacle to access the low abundant proteins (LAP), where the majority of biologically relevant and clinically important biomarkers reside. Therefore, isolation of MAP is a new challenge for effective detection and analysis of LAPs. To tackle this challenge, we further developed the IgY-microbead system by immunizing chickens with a flow-through fraction of IgY12 column and constructing the column with affinity-purified IgY antibodies against the



flow-through proteins of IgY12 column. The column devel-oped, called SuperMix, was applied for further partition-ing of the flow-through fraction of IgY12, which resulted in a bound/eluted fraction (designated as MAP fraction) and the flow-through fraction (designated as LAP fraction). Unfractionated and serial-fractionated samples using IgY12 and SuperMix columns were analyzed by SDS-PAGE and 2DE. Our data demonstrate that SuperMix columns specif-ically and reproducibly remove the post-IgY12 layer of the abundant proteins. A case study using SDS-PAGE coupled with LC/MS/MS demonstrates that the SuperMix column enabled specific capturing of 207 MAP, with 77 proteins being uniquely identified in high confidence (≥95%). This novel approach enables deeper and more effective access into the population of LAPs. In addition to digging deeper with the SuperMix column, we also have progressed in the direction of digging faster. One of the present challenges of plasma biomarker discovery is sample throughput limita-tions. In collaboration with PSS Bio Instruments, GenWay Biotech has developed a novel multiplex automated system (SepproTip) for high-throughput plasma sample processing. It permits processing of 12 samples at a time. This Seppro-Tip system can process plasma samples using both IgY12 and SuperMix tips. The turnaround time of 12 samples per 65 min allows a large number of samples to be processed without decrease in sample preparation quality.

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Sample Preparation for Body Fluid Profiling using Magnetic Bead TechnologyD. Gillooly1, J. Knol2, C. E. Teunissen3, M. Simmelink2,3, C. R. Jimenez2; 1Invitrogen Dynal AS, Oslo, Norway, 2OncoProteomics Laboratory, VUmc-Cancer Center, Amsterdam, The Netherlands, 3Dept. of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.

Peptide profiling of biological samples using MALDI-TOF mass spectrometry (MS) is an increasingly popular approach used for the discovery of biomarkers and as a means to detect and diagnose disease and allow the assess-ment of disease severity, progression, and the effectiveness of treatments.

Before mass spectrometry can be used to generate body fluid peptide profiles, reproducible, preferably automated, sample preparation procedures need to be used in which peptides are enriched and substances which interfere with MS analysis removed. Dynabeads are uniform superpara-magnetic monodisperse beads with a specific and defined surface for the adsorption/desorption and coupling of bio-reactive molecules. We have developed ion exchange and reversed-phase magnetic beads for peptide isolation from body fluids. These beads enable large numbers of peptides to be processed and analyzed simultaneously. The use of multiple bead surfaces for the capture of peptides from the same sample increases the number of peptides detected in a given sample and may increase the likelihood of discover-ing novel biomarkers. Sample preparation procedures have been successfully automated on robotic platforms.

Using peptide capture automated on the KingFisher96 coupled to MALDI-TOF-MS read-out, we investigated pre-

analytical variables and have identified peptides sensitive to differences in clotting time. Furthermore, we have opti-mized the protocol for capture of native CSF peptides.

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Mass Spectrometric Analysis of Metals for Biomarker Identification and Confirmation in Biological Model SystemsW. Johnson, Jr., L. Dimico, B. Buckley; EOHSI, Piscataway, NJ, United States.

Metals are potentially good biomarkers because they cannot be created or destroyed the way organic analytes can. They are frequently an integral part of many biological systems, such as protein binding. One property of metals is their ability to act as a label for small-molecule metabolism. Specifically, many small organometallic molecules can be measured in a biological compartment using the metal as the label.

Mancozeb (MZ), a fungicide comprised of an organic backbone and a metal moiety in a 9:1 ratio of Mn to Zn, is neurotoxic. Its metabolite, ethylene thiourea (ETU), is a known teratogen, carcinogen, and anti-thyroid com-pound. It is one molecule that has both an organic moiety and a metal analyte. Measuring the organic backbone is often more difficult than measuring the metal because the organic moiety appears to be unstable. Measurement of MZ in biological matrixes and demonstration of its uptake by neuronal cells proved difficult by conventional LC/MS.

Quantification by liquid chromatography/mass spec-trometry of the organic analyte inside the neuronal cells showed that about 8% of the compound crossed the cell membrane. This was significant because no one had mea-sured MZ inside the cell, even though its neurodegenera-tive properties were known. The results were not com-pletely conclusive because the precursor ion could not be measured inside the cell, only product ions. ICP/MS was used to measure the Mn content inside the cell and discover that about 8% of the MZ had entered the cell. The agree-ment between the two methods was significant and dem-onstrated the utility of the metal as a confirmatory analysis. It also confirmed that the Mn had entered the cell and not just the organic backbone. This presentation will focus on the use of metals as labels in biological systems, including analytical methods and stable isotope labeling.

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Identification of Protein Biomarkers Associated with Hypoxia in Human Malignant Glioma Cell Lines Using Proteomics TechnologiesT. Kempf1, S. Rahn1, U. Warnken1, M. Schnoelzer1, W. Wick2; 1German Cancer Research Center, Heidelberg, Germany, 2University of Tuebingen Medical School, Tuebingen, Germany.

The most common type of primary malignant brain tumors are glioblastomas. These highly aggressive, rapidly growing tumors are exposed to hypoxia, which occurs as a consequence of inadequate blood supply. Hypoxia exerts a



variety of influences on tumor cell biology. Among these are activation of signal transduction pathways, adapting hypoxic tumor cells to an anaerobic environment.

As a complementary approach to gene expression pro-filing, we aimed to investigate changes in the overall pro-tein pattern of malignant glioma cell lines after hypoxia treatment compared to normoxic controls utilizing various proteomics techniques.

The human malignant glioma cell line LNT-229 was initially used in our studies. Three individual samples rep-resenting three cell cultures grown under identical condi-tions were taken at four different time-points of hypoxia treatment (control, 8, 24 and 72 h). Proteins from total cell lysates were separated by high-resolution 2D gel elec-trophoresis and visualized by silver staining. Computer-assisted image analysis of the gels allowed the detection of differentially expressed proteins.

Proteins spots that were recognized to be up- or down-regulated were identified by mass spectrometry in prepara-tive gels. We have found 14 proteins up-regulated and 6 proteins down-regulated in hypoxia compared to normoxia based on the image analysis of the corresponding 2D gels. These results are currently being verified by Western-blot analysis of samples from three different glioma cell lines—LN-18, U87, and LNT229. Furthermore, all candidate proteins will be evaluated on primary cell cultures from human gliomas, and immunohistochemically on glioma tissue microarrays.

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HT Proteomics LC/MS For Analysis of Low-Abundance Proteins, PTMs, and BiomarkersK. Nugent, P. Kent, L. Upton; Michrom Bioresources, Auburn, CA, United States.

The introduction of electrospray ionization (ESI) in the 1980s revolutionized the analysis of biomolecules, and LC-ESI/MS (50–5000 µL/min) offers robust, high-throughput analyses for many biological applications. The introduction of nanospray (nESI) in the 1990s has made nLC-nESI/MS (10–1000 nL/min) a valuable tool for pro-teomics research, where high resolution and high sensitivity are maximized at the cost of sample throughput (60–240+ min per analysis) and robustness. Although protein bio-marker analysis requires the high resolution and high sen-sitivity provided by nanoLC/MS, it also requires robustness and high throughput if it is to be useful in pharmaceutical and clinical applications. This study introduces a new axial desolvation, vacuum-assisted nano-capillary electrospray (ADVANCE) source for LC/MS, which has the robustness of LC-ESI/MS and the sensitivity of nanoLC-nanoESI/MS, but operates in the flow range from 0.2–100 µL/min. For low-abundance proteins, PTMs, and biomarker identifica-tion, an HT capLC-ADVANCE/MS system can analyze 50–150 host cell proteomic samples (digests of 1D gel slices, 2D gel spots, or MDLC fractions) in 24 h (10–30 min per analysis) at attomole to picomole sensitivities. The capLC-ADVANCE/MS system can also be used for validation of biomarker candidates, which requires comparative quan-titation (ICAT, ITRAC, SILAC) of proteomic samples (5–15 min per analysis) in host cells and physiological

fluids. Once protein biomarkers have been validated (very few to date), the capLC-ADVANCE/MS can be used for very high throughput (2–5 min per analysis) quantitation of biomarker signature peptides in a variety of diagnostic and therapeutic applications.

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Streamlining Plant Sample Preparation: The Use of High-Throughput Robotics to Process Echinacea Samples for Biomarker Profiling by MALDI-TOF Mass SpectrometryS. A. Schwartz1, I. Issac2, L. A. Greene1, J. R. Guthrie1, D. Gray1; 1Midwest Research Institute, Kansas City, MO, United States, 2Genomic Solutions, Ann Arbor, MI, United States.

Several species in the genus Echinacea are beneficial herbs popularly used for many ailments. The most popu-lar Echinacea species for cultivation, wild collection, and the development of herbal products include E. purpurea (L.) Moench, E. pallida (Nutt.) Nutt., and E. augustifolia (DC). Product adulteration is a key concern for the natural products industry, where botanical misidentification and the potential for introduction of other botanical and non-botanical contaminants exist throughout the formulation and production process. Therefore, rapid and cost-effective methods that can be used to monitor these materials for complex product purity and consistency are of benefit to consumers and producers. The objective of this continu-ing research was to develop automated, high-throughput methods to process samples and differentiate Echinacea spe-cies by their MALDI-TOF mass profiles. Based on analysis of pure Echinacea samples, off-the-shelf products contain-ing Echinacea could then be evaluated in a streamlined process.

Leaf/flower pieces, seeds, and roots from E. purpurea and E. augustifolia; seeds and roots from E. pallida; and off-the shelf Echinacea supplements were extracted in Tris buffer using bead-beating technology. Samples were then transferred, diluted, and deposited on a MALDI tar-get plate for MS analysis using customized methods on a ProPrep liquid-handling system (Genomic Solutions). MS analysis of these samples highlighted key MS signal pat-terns from both small molecules and proteins that char-acterized the individual Echinacea samples analyzed. Cor-responding analysis of dietary supplements was used to monitor Echinacea sample composition, including species and plant material used. These results highlight the poten-tial for streamlined, automated approaches for agricultural species differentiation and botanical product evaluation.

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The Effect of Brief Exercise on Soluble Adhesion Molecules and Soluble Selectins in Early and Late Pubertal MalesC. D. Schwindt; UCI, Orange, CA, United States.

It now appears that many of the health effects of exer-cise are influenced by the balance of stress mediators and


JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007 1�

growth factors. Related to these agents are the soluble adhe-sion molecules (sAM: ICAM and VCAM) and soluble selectins (sS: E, L, and P selectins) which have been linked to such illnesses as asthma and cardiovascular disease. We hypothesized that brief exercise would alter circulating lev-els of sAM and sS, and that the response would be modified by the child’s pubertal status. Thirty healthy males (14 early pubertal, EP; 16 late pubertal, LP) performed 10 2-min bouts of exercise on a cycle ergometer. Blood was sampled at pre-exercise (PE) and end-exercise (EE). Levels of sAM and sS were analyzed using commercially available ELISAs. Mean PE levels were significantly different between groups for ICAM (p < 0.005) and VCAM (p < 0.05), and at EE for ICAM (p < 0.005). VCAM significantly increased in response to exercise in LP males (p < 0.05). A significant change between groups was demonstrated for the selec-tins at PE (E and P p < 0.005; L p < 0.05) and EE (all sS p < 0.05). Only LP males demonstrated a significant change from PE to EE (all sS p < 0.05). Baseline levels of circulat-ing vascular adhesion molecules are significantly different between healthy EP and LP males. Moreover, exercise in LP males caused a significant increase in these vascular media-tors, possibly preparing the organism to adapt to stressors imposed by exercise and the vascular demands needed for muscle growth. NIH- P01HD048721, RO1HL080947, K23 ES014923-02.

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Quantitative Measurement of Gene Expression from Formalin-Fixed Tissue Providing Identical Results as Fresh Tissue: Biomarker Validation using Clinical SamplesB. Seligmann1, L. Rimsza2, R. Martel1, C. Sabalos1, R. Robin3, I. Botros1, M. Rounseville1, M. LeBlanc4, J. Unger4, T. M2, T. Grogan2; 1HTG, Tucson, AZ, United States, 2University of Arizona, Tucson, AZ, United States, 3University of Arizona, Visiting Scientist, Tucson, AZ, United States, 4Fred Hutchinson Cancer Research Center, Seattle, WA, United States.

The measurement of gene expression from formalin-fixed paraffin-embedded (FFPE) tissue has proven to be problematic. Consequently, archives of FFPE samples remain unexploited in the quest to elucidate and validate the molecular mechanisms of diseases, cellular processes, and drug activity/safety. We validated the measurement of gene expression from FFPE tissue by a new multiplexed assay, the quantitative nuclease protection assay (qNPA). qNPA measures the RNA cross-linked to tissue without its having to be solubilized, plus the soluble RNA pool—i.e., the total RNA contained in the FFPE sample. This is likely one explanation of qNPA success where methods that measure only soluble RNA have failed. Cross-linked RNA is the major pool in FPE samples, and the fraction it makes up varies from sample to sample, presumably due to differences in fixation time or sample age. Consistent with the observation that qNPA measures the total RNA in fixed tissue, and the fact that it measures the total RNA in fresh samples, is the result that identical quantitative levels of gene expression are measured from fresh fixed and 18-y-old FFPE tissue and from matched fresh or fixed

tissue. The expression level for a set of low to moderately expressed genes from fresh vs. FFPE tissue correlated with an R2 = 0.99, slope = 1. Gene expression measurements in FFPE tissue provided average CVs <10%. A retrospective study using clinical diffuse large B-cell lymphoma samples was carried out, validating prognostic biomarkers of disease, disease subtype, and survival. The levels of gene expression measured by qNPA correlated with protein product levels measured by IHC. These results validate that qNPA pro-vides a high-quality gene expression assay of FFPE tissue, enabling research and clinical assays not previously pos-sible.

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Characterization of the Analytical Performance of an FT-MS-Based Label-Free Platform for Biomarker Discovery in Human Plasma: Standards, Precision, Accuracy, and Directed Feature Investigation Using SIEVEJ. N. Sutton1, M. Athanas2, A. Prakash1, R. Gerszten3, L. Bonilla1; 1Thermo Fisher Scientific, Cambridge, MA, United States, 2The BioTeam, Cambridge, MA, United States, 3Massachusetts General Hospital, Boston, MA, United States.

In this report we present a detailed methodological and instrumental description of an FT-MS-based, label-free platform for biomarker discovery in human plasma. We will discuss the relevant analytical figures of merit for each of the main steps of this workflow, namely immunoaffinity depletion of the 12 most abundant plasma proteins; high-precision and high-resolution LC-MS/MS analysis using a unique split-flow design; robust differential MS signal analysis using a robust high-throughput computational assembly that employs new chromatographic alignment and time-course statistics-based differential expression algorithms (SIEVE), followed by directed feature (frame) identification using Sequest. The application of this analyt-ical platform to a relevant clinical model will be discussed, as well as its extension to targeted feature analysis by com-plementary MS strategies (ECD, IRMPD), and potential new applications such as metabolomics.

carBohydr ate analysis

P37-S

Glycan Analysis of Prion Proteins (PrPc) by Mass SpectromeryP. Azadi1, M. Ishihara1, C. Li2; 1University of Georgia, Athens, GA, United States, 2Case Western Reserve University School of Medicine, Cleveland, OH, United States.

Prion diseases are a class of neurodegenerative diseases in which conversion of a normal cellular glycoprotein, the prion protein (PrPc), to a misfolded form (PrPSc) generates a protein that is believed to be the sole component of the


1� JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007

transmissible agent of the diseases. It is the accumulation of PrPSc in the CNS that causes neurodegeneration and gliosis, which are characteristic of the diseases. Conforma-tional changes in PrPc that lead to PrPSc cause the protein to become proteinase resistant and to form fibrils.

PrPc is a glycoprotein expressed by many cells, includ-ing neurones and muscle cells. However, it is predominantly expressed at synapses, suggesting that, functionally, PrPc is important for neuronal activity. The fusion glycoprotein was expressed in CHO cells and purified with protein G beads.

The aim of this study has been to characterize the N-linked oligosaccharides on the prion protein. The released and permethylated N-linked oligosaccharides were ana-lyzed by both MALDI-MS and ESI-MS. The structure of N-linked glycans was confirmed by MS/MS analysis of permethylated glycans.

The N-linked profiling experiment produced a compli-cated set of data with more than twenty different oligosac-charides that are present on this glycoprotein. The main structures were the fucosylated biantennary structure, with one galactose, and the fucosylated biantennary structure, with two galactose residues. Larger triantennary and tetra-antennary structures were also detected. The structure of different forms of prion proteins will be investigated.

P38-M

Improved Characterization of Glycopeptides Using CID and ETD in a Non-Linear Ion Trap MSC. Baessmann1, M. Wuhrer2, M. Lubeck1, R. Hartmer1, A. Brekenfeld1, C. A. M. Koeleman2, A. M. Deelder2; 1Bruker Daltonik GmbH, Bremen, Germany, 2Leiden University Medical Center, Leiden, The Netherlands.

Characterization of glycoproteins by electrospray ion-ization tandem mass spectrometry of glycosylated peptides using collisionally induced dissociation (CID) results in the preferential fragmentation of the glycosidic bonds, while the peptide bonds are more stable. As low-energy CID of glycopeptides mainly provides information on the gly-can moiety, a selective analysis of the peptide chain itself usually requires enzymatic removal of the carbohydrate part. The recently introduced electron transfer dissocia-tion (ETD) option for ion-trap mass spectrometers shows preferred fragmentation of the peptide backbone, leaving posttranslational modifications widely intact. Thus, a com-bination of ETD and CID is a highly promising tool for the analysis of glycopeptides.

A high-capacity three-dimensional quadrupole ion-trap MS with ETD option enables the application of the two complementary fragmentation techniques. For fragmenta-tion via ETD, radical anions of fluoranthene are gener-ated in a negative chemical ionization source and added to previously isolated multiply charged peptide cations inside the ion trap. Different glycosylated proteins were analyzed either offline or with on-line nano-LC-MS/MS in the posi-tive-ion mode. Enriched glycopeptides from a tryptic digest of horseradish peroxidase were analyzed offline using nano-spray. For an on-line nano LC approach, digests of human IgG3 were chosen.

With ETD and CID, widely complementary data from these glycopeptides were obtained: CID MS/MS spectra showed mainly sugar fragmentation, while ETD MS/MS allowed for selective fragmentation of the peptide back-bones with retention of the intact glycan moiety, thereby allowing the facile deduction of peptide sequence and gly-can attachment site. In conclusion, glycopeptides were char-acterized in detail with regard to carbohydrate composition and structure as well as peptide sequence and modification sites. Importantly, this approach can be incorporated into a conventional glycoproteomics work flow, as sensitivity and speed of data acquisition allow the combined use of ETD and CID with direct LC coupling.

P39-T

Analysis of Oligosaccharides by Capillary-Scale High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (CHPAEC-PAD) and On-Line Electrospray-Ionization Ion-Trap Mass Spectrometry (CHPAEC-ITMS)C. Bruggink1,2, C. Koeleman2, V. Barreto3, Y. Lui3, C. Pohl3, A. Ingendoh4, M. Wuhrer2, C. Hokke2, A. Deelder2; 1Dionex, Amsterdam, The Netherlands, 2Leiden University Medical Center, Dept. of Parasitology, Leiden, The Netherlands, 3Dionex Corp., Sunnyvale, CA, United States, 4Bruker Daltonics GmbH, Bremen, Germany.

High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is an established technique for selective separation and analysis of underi-vatized carbohydrates. The miniaturization of chromato-graphic techniques by means of capillary columns, and on-line coupling to mass spectrometry are critical to the further development of glycan analysis methods that are compatible with the current requirements in clinical set-tings.

A system has been developed based on the Dionex BioLC equipped with a microbore gradient pump with PEEK flow splitter, a FAMOS micro autosampler, a modi-fied electrochemical cell for on-line capillary PAD, and a capillary column (380 µm i.d.) packed with a new type of anion-exchange resin. This system operates with sensitivity in the low femtomol range. In addition, an on-line capillary desalter has been developed to allow direct coupling to the Bruker Esquire 3000 ion-trap mass spectrometer with elec-trospray ionization interface (ESI-IT-MS). Both systems have been evaluated using oligosaccharide standards as well as urine samples exhibiting various lysosomal oligosaccha-ride storage diseases. Initial data indicate that the robust and selective anion-exchange system, in combination with ESI-IT-MS for structure confirmation and analysis, pro-vides a powerful platform that complements existing nano/capillary LC-MS methods for analytical determination of oligosaccharides in biological matrices.



P40-S

Isobaric Labeling for Comparative GlycomicsR. Orlando, J. A. Atwood, III, L. Cheng, W. S. York; CCRC/UGA, Athens, GA, United States.

A novel strategy, based on the use of isobaric labeling, is introduced for quantitative glycomics. In this approach, glycans are permethylated with either 13CH3I or 12CH2DI. This pair of reagents has the same nominal mass but differ in their exact mass by 0.002922 Da per label. This mass difference is difficult to resolve with current mass spec-trometers in cases where only a single label is attached to the analyte. However, glycans typically contain mul-tiple hydroxyl groups, which increase the mass difference between the two samples. Since the number of hydroxyl groups increases with the mass of the glycan, the difference between these isobaric species also increases and thus the resolution needed is approximately 25,000 ΔM/M and is independent of the glycan’s size for typical N- and O-linked species. The advantages of this labeling strategy are numer-ous, and result primarily from the isobaric ions appearing at the same nominal mass-to-charge ratio. This characteristic leads to increased ion intensity as ions from both samples are not distributed between isotopic species having differ-ent m/z values. The small mass difference between these isobars allows the two species to be simultaneously selected for MSn analysis, permitting the relative quantitation of isomeric glycans. This procedure was successfully used to analyze N- and O-linked oligosaccharides released from a standard glycoprotein and from human serum. This strat-egy is directly applicable to oligosaccharides from other sources, such as glycolipds. The concept of isobaric label-ing is expected to be applicable to other types of “omics” analyses with other derivatizing agents.

P41-M

Automated LC-MALDI Analysis of Glycopeptides from Glycoprotein Digests Using DHB as MatrixA. Resemann1, A. Asperger1, K. Sparbier1, K. Seemann2, T. Eichhorn2, C. Hunzinger2, G. Stein1, L. Vorwerg1, D. Suckau1; 1Bruker Daltonik GmbH, Bremen, Germany, 2Merck KGaA, Darmstadt, Germany.

2,5-Dihydroxybenzoic acid (DHB) is the matrix of choice for carbohydrate and glycopeptide analysis, but due to the inhomogeneous surface morphology of samples pre-pared with DHB, it is typically incompatible with auto-mated measurements.

We describe a simple and rapid method for the analysis of glycoproteins, which combines (a) reducing the complex-ity of the digest mixtures with glyco-specific enrichment and (b) subsequent LC-MALDI-TOF MS/MS analysis with DHB as MALDI matrix. All samples were prepared on hydrophobic sample plates with hydrophilic anchors 400 or 600 µm in diameter confining the sample dimen-sions.

In a first step, the matrix was applied to the 384 sample spots (“anchors”) of a microtiter plate–shaped MALDI target. The LC eluate from CAP-RP-HPLC subsequently dissolved the DHB matrix confined to the hydrophobic boundaries of the anchors. Co-crystallization of glyco-

peptides in DHB suitable for the automated analysis was achieved.

This method was applied to recombinant human inte-grin alpha and beta; glycosylation sites were identified and described.

The MALDI-MSMS spectra of glycopeptides (N-linked type) include information about the structure of peptide moiety as well as the glycan part of the molecules. MALDI-TOF/TOF spectra permitted (a) the detection of N-linked glycopeptides by a neutral loss analysis across the entire LC-MALDI-MS/MS dataset, (b) the determination of the molecular weight of the pure peptide chain by typical fragmentation patterns, (c) the identification of the peptide part of the fragmented glycopeptide by means of simple database searching, and (d) initial information about the glycan composition and the attachment site.

LC-MALDI-TOF/TOF on DHB matrix preparation is a powerful approach for the detailed characterization of glycoproteins.

P42-T

Analysis of Complex Carbohydrates Using a Hybrid Ion Trap and TOF Mass Spectrometer Coupling with MALDIF. Xiang1, J. Zhao2, D. M. Lubman2; 1Shimadzu Biotech, Pleasanton, CA, United States, 2The University of Michigan, Ann Arbor, MI, United States.

Living systems are dynamic, each developing, surviv-ing, and proliferating in a different way. A very important element in understanding system dynamics is to quantify change. Quantitative measurement of proteins is therefore increasingly requested by life science researchers as a means of characterizing complex biological systems, including human cells, tissues, and body fluids. Mass spectrometry has became a major tool for proteomics applications, and allowed the quantitative measurement of protein expres-sion in many biological systems when combined with stable isotope coding. This presentation shows an approach to quantification and identification in proteomics applica-tions, for the first time using a hybrid ion trap and TOF mass spectrometer combined with stable isotope coding. The mass spectrometer is equipped with LC MALDI to meet separation needs, in-source decay (ISD), and collision-induced dissociation (CID) to generate MSn spectra. The quantification is achieved in two ways. Peptide mass shift resulting from the stable isotope coding can be measured directly in MS mode, and relative abundance of coded and non-coded peptides is read out from the MS spectra. The samples coded with the tandem mass tags (TMTs)1 strategy are analyzed with ISD or MSn mode, and then the relative abundance of the reporter ions from TMTs are measured for the quantification. Metabolic incorporation of stable isotope (15N) labeled nutrients in growth media into cul-tured cells is used for global coding of proteomes. Labeled and non-labeled proteins were separated and purified from cell lysates, tryptically digested, and then mixed in differ-ent ratios for MS-mode measurement. Standard proteins labeled with TMTs were used for ISD and MSn-mode measurement. The results demonstrate that high signal-to-noise ratio is achieved and both the identity and relative



abundances are obtained simultaneously with MSn-based detection; PTM information could be obtained as well.

computational Biology

P43-S

Computational Biology Applications Suite for High-Performance Computing (BioHPC.net)J. Pillardy; Cornell University, Ithaca, NY, United States.

One of the challenges of high-performance computing (HPC) is user accessibility. At the Cornell University Com-putational Biology Service Unit, which is also a Microsoft HPC institute, we have developed a computational biology application suite that allows researchers from biological lab-oratories to submit their jobs to the parallel cluster through an easy-to-use Web interface. Through this system, we are providing users with popular bioinformatics tools includ-ing BLAST, HMMER, InterproScan, and MrBayes. The system is flexible and can be easily customized to include other software. It is also scalable; the installation on our servers currently processes approximately 8500 job submis-sions per year, many of them requiring massively paral-lel computations. It also has a built-in user management system, which can limit software and/or database access to specified users. TAIR, the major database of the plant model organism Arabidopsis, and SGN, the international tomato genome database, are both using our system for storage and data analysis.

The system consists of a Web server running the inter-face (ASP.NET C#), Microsoft SQL server (ADO.NET), compute cluster running Microsoft Windows, ftp server, and file server. Users can interact with their jobs and data via a Web browser, ftp, or e-mail. The interface is accessible at http://cbsuapps.tc.cornell.edu/.

P44-M

Calculation of the Isoelectric Point of Tryptic Peptides Based on Adjacent Amino Acid EffectsJ. R. Sevinsky1, B. J. Cargile1, A. S. Essader1, J. P. Eu2, J. L. Stephenson, Jr.1; 1Research Triangle Institute, Research Triangle Park, NC, United States, 2Duke University Medical Center, Durham, NC, United States.

Current algorithms for the calculation of peptide or pro-tein pI, based solely on the charge associated with individual amino acids, can calculate pI values to within ±0.2 pI units. Here we present a new pI calculation algorithm that takes into account the effect of adjacent amino acids on the pI value. The algorithm takes into account the effect of adja-cent amino acids ±3 residues away from a charged aspartic or glutamic acid, as well as effects on the C-terminus, and applies a correction factor to the pK values of the charged amino acids. Large pK shifts are observed for the short-range interactions of aspartic and glutamic acid with the N-terminus and with internal histidine, lysine, or arginine residues. Conversely, interactions of aspartic and glutamic

acid with hydrophobic, slightly polar, or other aspartic and glutamic acid residues are negligible. This results in a much narrower distribution of peptides across individual IPG-IEF fractions. The correction factors are derived from a 5000-peptide training set using a genetic optimization approach. The unique advantage of the genetic algorithms is that the pK optimization problem is independent of the evaluation function employed. This in turn allows the optimization function to determine the next set of solu-tions that can then be re-evaluated. This cycle continues until a set convergence criterion is reached. The accuracy of the new pI values obtained with this method approaches the error associated with the manufacture of the IPG strip (±0.05 pI units). The approach is demonstrated for cyto-solic cell extracts derived from the breast cancer cell line DU4475, and from membrane preparations from human lung tissue samples.

dna sequencing

P45-T

Evaluation of a Novel Surface-Modified Microplate for Sequencing Reaction PurificationJ. O. Adigun, A. R. Bennet, O. Zimerman; Edge BioSystems, Inc., Gaithersburg, MD, United States.

Sequencing reaction purification (SRP) is typically performed using gel filtration, solid magnetic substrate, membrane platform, or standard ethanol precipitation. The samples are centrifuged, magnetically separated, vacuum filtered, or precipitated and centrifuged, respectively, in order to eliminate salts, unreacted dNTPs, and dye termi-nators from the extension products. We have developed a surface-modified microplate to perform SRP without the need for these manipulations.

With this technology, the extension products are selec-tively adsorbed to the surface-modified multi-well plate, washed with alcohol, and eluted in water. The system was evaluated in 96- and 384-well plate configurations on an ABI 3730XL DNA Analyzer using the BigDye v3.1 chem-istry. Results were obtained with typical Phred20 scores of 800.

The plates performed reproducibly over a wide range of reaction conditions. The system was tested using sequenc-ing reaction volumes ranging from 5 to 10 µL, DNA tem-plate masses ranging from 10 to 100 ng, and BigDye vol-umes ranging from 125 nL to 2 µL. Results were obtained both manually and via automation.

With no adjustment to the protocol, the modified plate system was very robust to variations in template and BigDye

concentration as well as sample volume. Using a Biomek FX workstation, plate processing times were less than 30 min. Thus, this new technology offers a robust, high-capacity, and fully automatable dye terminator removal platform to work efficiently with reduced volumes of dye terminators.



P46-S

A New Method for the Purification of DNA Sequencing ReactionsG. Amparo, M. Harrold, S. Pistacchi; Applied Biosystems, Foster City, CA, United States.

BigDye XTerminator is a new single-tube method for purifying DNA sequencing reactions prior to electropho-retic analysis. Sequencing reactions purified using this method display very few artifacts from residual dye-labeled nucleotides (dye blobs), as well as excellent recovery of the smallest extension products. BigDye XTerminator also has unique workflow advantages over conventional purifica-tion schemes such as ethanol precipitation or spin column purifications.

BigDye XTerminator is added directly to finished DNA sequencing reactions and mixed. After mixing, the samples purified with BigDye XTerminator can be directly injected from the reaction tube without additional transfer steps. Performance of BigDye XTerminator with a variety of DNA sequencing reactions will be demonstrated. Work-flow and automation issues will also be described.

P47-M

Comparison of Pyrosequencing and Fluorescent Dideoxynucleotide Sequencing for Measurement of CpG MethylationK. Hager; Yale U., New Haven, CT, United States.

In mammals, cytosine methylation at CpG sites is an important epigenetic mechanism for altering gene expres-sion in normal and cancer cells. Sequencing of PCR prod-ucts from bisulfite-treated genomic DNA provides the most direct measurement of methylation at individual cytosines. To compare the relative ability of pyrosequencing and fluo-rescent dideoxynucleotide sequencing to accurately quan-tify cytosine methylation, I designed primers for amplifica-tion of a 197-bp region within the IGF2 gene. Two sets of primers were synthesized: (1) a set containing a 5′-biotin on the reverse primer (used for streptavidin purification of the template strand prior to pyrosequencing) and (2) a set containing the M13 forward and M13 reverse primer sequences as 5′ extensions of the forward and reverse PCR primers, respectively. Only the first set was used to gener-ate PCR products for pyrosequencing; both sets were used to make amplicons for fluorescent DNA sequencing. Fol-lowing PCR amplification of eight bisulfite-treated human genomic DNAs, templates were purified and subjected to both DNA sequencing technologies. Pyrosequencing was able to measure percent methylation at 7 CpG sites, while the fluorescent chemistry was able to interrogate up to 12 sites, due in part to its longer read length. The percent methylation measured by each technology differed for a given CpG site and genomic DNA: the value obtained by pyrosequencing was usually 10–15% less than that obtained by the fluorescent chemistry. In order to determine which chemistry measures percent methylation more accurately, experiments with known ratios of fully methylated and unmethylated templates are underway.

P48-T

A New High-Density Size Standard for Sizing Large Fragments Across Multiple Fragment Analysis Capillary Electrophoresis ApplicationsS. Karudapuram, R. Padilla, S. Chen, S. Koepf, J. Hauser, Y. Wang, K. Jacobson, M. White, R. Bordoni, M. Wenz, A. Shah, L. Joe; Applied Biosystems, Foster City, CA, United States.

When performing large-fragment analysis applica-tions on capillary electrophoresis, a high-performance size standard is essential. Applications such as AFLP, T-RFLP, VNTR, mutation screening, and BAC fingerprinting com-monly generate fragments greater than 600 base pairs. Using the Applied Biosystems 5th dye technology, we have developed a new high-density size standard containing over 65 peaks, spaced no more than 40 base pairs apart. This new size standard provides accurate and precise fragment sizing from 20 to1200 base pairs, allowing the flexibility to analyze small and large fragments. We present details about the size standard and illustrate its versatility, accu-racy, and precision with a variety of large-fragment analysis applications.

P49-S

Recent Improvements in SOLiD Sequencing ChemistryB. Nutter; Applied Biosystems, Foster City, CA, United States.

The sequencing by oligonucleotide ligation and detec-tion (SOLiD) system being commercialized by Applied Biosystems generates sequences of randomly generated fragment or mate-paired DNA molecules utilizing a novel ligation-based chemistry and fluorescent probe pools. Con-sistent improvements have been made in total sequence throughput per run by enhanced read length and base calls, higher bead densities per array, and increased frequency of assignable reads. In combination, these improvements have allowed progressively deeper sequencing coverage of more complex model organisms. To date, we have sequenced, to varying depths of coverage, the genomes of several bacte-rial genomes. In addition to whole genome sequencing, we have developed a workflow to allow targeted resequencing and are working with collaborators to develop tag-based sequencing applications. Improvements to the SOLiD sequencing technology and the impact on the increased sequencing throughput as they apply to a number of differ-ent applications will be presented.

P50-M

High-Throughput and Automatic Plasmid DNA Preparation with SeqPrep TechnologyG. Wang; Edge BioSystems, Inc., Gaithersburg, MD, United States.

SeqPrep technology is developed for easy and quick DNA preparation from bacterial culture. It consists of a surface-modified microplate and a cell lysis/DNA-binding



solution. The DNA binds to the surface while cells get lysed. With this technology, DNA preparation is not only highly automatable but also very easy manually. Several robotic plaforms have been validated for plasmid DNA prepara-tion with SeqPrep. Data will be presented. Optimization for individual labs’ needs will be discussed in four aspects of the preparation process: bacterial culture, cell harvest, DNA isolation and DNA elution.

P51-T

Optimizing Heat Lysis for High-Throughput Sequencing of Various Plasmid LibrariesC. L. Wright, L. Hetrick, E. J. Campos, E. Vlach, A. Reese, A. G. Hernandez, R. W. Kim; University of Illinois, Urbana, IL, United States.

Heat lysis is a very reliable method for high-throughput labs to decrease costs and increase throughput with little sacrifice in quality of sequence data compared to typically used two-plate or magnetic-bead DNA purification meth-ods. Laboratories employing this procedure on high-copy plasmids, using a sequencing reaction with a 1/8th Big Dye (Applied Biosystems) dilution, have reported generating data with 85% of all wells having read lengths of at least 600 bases with quality value (QV) 20 or above.1

We have adopted this procedure in our laboratory and optimized it for sequencing from various in-house cDNA and genomic shotgun libraries cloned into high-copy plas-mids, as well as for libraries constructed outside of our sequencing center. To fully develop the procedure, we utilized a number of culture plates, testing different plate volumes, well shapes, and growth times. Also, we tested various volumes of resuspension buffer in order to gener-ate the highest sequencing success rates and read lengths. To further cut costs, we optimized the sequencing reac-tions by testing various dilutions of Big Dye and template amounts.

Based upon over 55,000 reads, we have been able to consistently generate sequencing results with average suc-cess rates of 90–95% and read lengths of over 700 bases with QV 20 or above. Our past protocol employed a stan-dard two-plate DNA preparation method with a 1/16th Big Dye dilution in the sequencing reaction. In contrast, we now use the optimized heat lysis protocol combined with a 1/32nd Big Dye dilution. These changes have increased throughput and produced the high-quality sequencing results stated above, yet reduced our consumables cost by over 55%.

reference

1. Ganguly T, Chen P, Teetsel R, Zhang LP, Papaioannou E, Cianciarulo. High-throughput sequencing of high copy num-ber plasmids from bacterial cultures by heat lysis. Biotechniques 2005;39:304–308.

P52-S

Characterization of the Number of CAG repeats in the Huntingtin Gene of a Huntington Disease (HD) Mouse Strain by Fragment Analysis and DNA SequencingM. R. Zianni, O. Glass, T. Ma, K. Hoyt; Ohio State University, Columbus, OH, United States.

Mutations in the human Huntingtin gene cause the neurological disorder Huntington’s disease (HD). Spe-cifically, the expansion of a trinucleotide repeat (CAG) in excess of 39 leads to a disease state in individuals. There-fore, determining the number of repeats in subjects is important, since the number of repeats has an effect on the severity of the pathology and the number of repeats shows a high degree of instability between generations. The pres-ent study utilizes fragment analysis and DNA sequencing to confirm the exact number of repeats present in exon 1 of the human Huntingtin gene in the transgenic mice used in a drug study. Since genotyping with an automated capil-lary electrophoresis instrument can be very precise, but not necessarily accurate as to the exact number of bases in the fragment, some of the PCR products were also sequenced. By the use of a 3730 DNA Analyzer, the actual fragment sizes as determined by DNA sequencing were found to be in close agreement with the predicted sizes based upon the fragment analysis—for example, 506 and 501 bases, respec-tively. The use of a mixture of sequencing kits, BigDye Ter-minator v3.1 and dGTP BigDye Terminator v3.0, without additives was found to be the best method to sequence over 100 trinucleotide repeats.

gene arr ays

P53-M

Real-Time PCR Array for Multi-Gene Expression Profiling and Microarray Data Validation: RT2

Profiler PCR ArrayE. Arikawa1, H. Pan1, Y. Sun1, J. Wang1, Q. Zhou1, S. Prabhakar1, Y. B. Wang1, S. Dial2, B. Ning2, L. Guo2, Y. Dragan2, J. Yang1; 1SuperArray Bioscience Corporation, Frederick, MD, United States, 2National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, United States.

Quantitative real-time reverse-transcription PCR (RT-qPCR) is presently the method of choice for validating gene expression results from high-density microarrays. However, the low throughput of traditional gene-by-gene RT-qPCR makes this process labor intensive and time consuming. To accelerate this laborious task, the SuperArray RT²Profiler PCR Array combines SYBR Green–based real-time RT-qPCR technology with a multi-gene array plate format to simultaneously analyze a panel of genes related to a specific disease or biological pathway.

Each assay on the PCR array plate has been experimen-tally validated to insure gene-specific amplification. The reliability and reproducibility of the RT²Profiler PCR Array have been demonstrated by DNA sequencing and intra/



inter-laboratory reproducibility comparisons. The DNA sequencing demonstrated 100% of the PCR products ampli-fied from the correct target genes. In one laboratory, indi-vidual PCR assays produced a standard deviation of 0.24 cycles and a coefficient of variance of 0.92% in technical replicates. The correlation coefficient for Ct values between replicate runs was 0.997 and for fold changes (ΔΔCt) across thermocyclers was 0.976. Comparisons between two dif-ferent laboratories using different thermocyclers showed correlation coefficients of 0.972 and 0.976 for ΔCt and ΔΔCt, respectively. Each PCR array also includes stringent controls to monitor RNA quality by assessing reverse tran-scription efficiency and genomic DNA contamination to ensure the reliability of the PCR array data.

A practical application for the PCR array was demon-strated by identifying human pancreatic tumor–associated genes using the Cancer PathwayFinder RT²Profiler PCR Array. Results showed 23 genes exhibiting a statistically sig-nificant threefold or greater change in expression between a human pancreatic tumor and normal pancreas, including many genes previously linked to pancreatic cancer. Hence, the RT2 Profiler PCR Array system offers a simple, reliable, and convenient tool for multi-gene profiling and micro-array data validation.

P55-S

Genomic Profile in AsthmaL. M. Teran, Dr.1, T. Ivacevic2, M. d. Garcia-Cruz1, V. Benes2; 1INER, Mexico City, Mexico, 2EMBL, Heidelberg, Germany.

Asthma is a complex genetic disease that is under the influence of many genes, and it has been proposed that different genes act in different families and different indi-viduals. The aim of the present study was to investigate at the genomic level the genes that may participate in the cau-sation and origins of asthma using microarray technology. A better understanding of these processes has the potential to identify markers of disease and new therapeutic targets. Seven atopic asthmatics and five non-asthmatics underwent bronchoscopy to obtain bronchial biopsies. Total RNA was purified from bronchial biopsies, and biotynilated comple-mentary RNA was prepared and hybridized to Affymetrix Hum 133 two plus chips (Affymetrix, Santa Clara CA). Using the multi-chip average procedure, we compared the mRNA expression profiles of 47,000 genes and/or EST sequences of atopic asthmatic with normal controls. Four hundred eighty genes had twofold mean regulation expres-sion differences or greater. Three hundred fifty-two genes were upregulated, including surface molecules involved in T- cell and B-cell activation, cytokines, extracellular matrix proteins, intracellular signaling products, and transcription factors. In conclusion, atopic asthmatics show a number of activated proinflamatory pathways that participate in T- and B-cell activation and airway remodeling. These differ-entially expressed genes identify potential molecular targets for preventive and therapeutic options in asthma.

genot yping

P56-M

High-Throughput Genotyping of International HapMap Project Populations with Applied Biosystems TaqMan Drug Metabolism Genotyping Assays: An Automated Laboratory and Analysis PipelineK. A. Haque, L. M. Wronka, C. L. Dagnall, C. M. Stefan, M. B. Beerman, B. D. Hicks, R. A. Welch, MS; National Cancer Institute, Division of Cancer Epidemiology and Genetics, SAIC-Frederick, Inc., Gaithersburg, MD, United States.

Although high-density whole-genome SNP scans are available for association studies, the tagging SNP approach used to design many of these panels from International HapMap Project data may miss a substantial number of coding functional variations of drug metabolism enzymes (DME). In fact, more than 40 DME genes are not covered by the HapMap Project, probably due to the difficulties in assay design for these highly homologous gene families. Additionally, many of these technologies do not provide detection in a high number of known DME genes, leading to further gaps in whole-genome scans. Of the polymor-phic putative functional DME variants not typed in Hap-Map, a large proportion is untagged by any combination of HapMap SNPs. Therefore, to correlate phenotypes to putative functional DME variations in pharmacogenomic studies, direct genotyping of these functional SNPs will be necessary. Applied Biosystems has developed a panel of N = 2394 TaqMan Drug Metabolism Genotyping Assays to interrogate putative functional variations in N = 220 DME genes. At the National Cancer Institute’s Core Genotyp-ing Facility, an automated, high-throughput pipeline has been created to genotype these assays on the International HapMap Project population. DNA sample preparation and handling, assay set-up, genotype analysis, and data publish-ing at SNP500 Cancer Database (http://snp500cancer.nci.nih.gov), have all been automated. Using a series of cus-tom-designed methods on five Beckman Coulter Biomek FXs, a Laboratory Information Management System, and analysis software, >650,000 genotypes have been obtained and analyzed by a single person in about 8 weeks. Using this pipeline, a completion rate of >99% and no Mendelian inheritance errors were observed. Furthermore, the CGF has implemented quality-controlled, automated pipelines for sample receiving, quantification, numerous DNA han-dling procedures, genotyping, and analysis for all samples and studies processed.



hplc of proteins and peptides

P57-T

Development of an Automated RP/RP 2D NanoLC/MS Method for Proteomic AnalysisH. Liu, G. Li, J. W. Finch, S. J. Geromanos, J. C. Gebler; Waters Corporation, Milford, MA, United States.

An automated RP/RP 2D nanoLC/MS method was developed and applied for E. coli digest analysis. Even though the first and second dimensions were both carried out with reversed phase (RP), the separation was performed with a high degree of orthogonality due to a sharp differ-ence in the pH of the mobile phases used for each dimen-sion (10 and 2.6, respectively). The automation of the 2D system was realized with the use of a novel on-line organic dilution method: when an organic-containing fraction was eluted from the RP column of the first dimension with pump 1, an aqueous flow was pumped into the system with pump 2 and mixed with the eluted fraction to reduce its organic content. When the fraction was collected with a RP trap column interfacing the two RP dimensions, the organic concentration in the fraction was lowered enough to allow the peptides present in the fraction to be retained. Then a nano-flow gradient was delivered from pump 2 through the trap column and the RP column of the second dimension for further separation.

The developed 2D method was successfully applied to sequencing a 500-ng E. coli global digest. The sample was fractionated 30 times with the first dimension. Each of the fractions was run with the second dimension using a 30-min gradient. There were more than 637 proteins iden-tified with a false-positive rate of 3.8%. There were 5171 peptides corresponding to the identified E. coli peptides, of which only about 10% were observed from two or more fractions, indicating a high fractionation quality. These data suggest that the RP/RP method may be preferable to the conventional SCX/RP for proteomic analysis.

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Optimization of Nanobore Capillary Trapping Column Geometries for Analysis of Peptide MixturesJ. M. Neveu1, C. J. Toher2, W. S. Lane1, G. A. Valaskovic2; 1Microchemistry and Proteomics Facility Harvard University, Cambridge, MA, United States, 2New Objective, Inc., Woburn, MA, United States.

Nanobore reversed-phase HPLC has become a main-stay for protein identification by modern tandem mass spectrometry. Enhanced robustness and versatility is obtained with trapping columns for on-line preconcen-tration and desalting of samples. Trapping columns of varying sizes and packing materials are available, yet few configurations are available for 75-µm i.d. packed-tip col-umns frequently employed in high-sensitivity work. Using a novel fluoropolymer and PEEK-based zero-dead-volume fitting with packed-tip, silica-fritted capillary columns, variable trap column geometries, and column chemis-

try combinations were investigated toward optimizing chromatographic performance in a typical nanobore RP-HPLC-MS system.

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Vydac MS RP-HPLC Columns Provide Unique Selectivity and High Recovery for Peptide and Protein SeparationsR. T. Nguyen, D. Chan; Grace Davison, Hesperia, CA, United States.

Based on specially treated large-pore silica and enhanced with a proprietary bonding process, Vydac MS reversed-phase (RP) high-performance liquid chromatography col-umns offer superior performance for peptides and proteins. The deamidation of human growth hormone (HGH) has been monitored for many years by RP using Vydac col-umns. The Vydac MS C4 column provides the best overall performance characteristics (recovery, resolution, and peak symmetry) for the common important assay of HGH and desamido HGH. Although hydrophobic membrane pro-teins are particularly difficult to separate, the Vydac MS C4 column provides better separation and recovery (up to 86% higher than other leading columns) for a reptilian reovirus p14 protein and myristolyated form, a component of a potentially new vaccine-delivery system. Separation of the trypsin digest of fetuin, a glycoprotein, exhibits improved selectivity for peptide mapping on a Vydac MS C18 column compared to other C18 columns, revealing some peaks otherwise not seen. The improved selectivity for peptides on the Vydac MS columns results in better primary structure definition and easier identification of degradation products and other protein characteristics.

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Rapid Peptide Analysis via Nanobore LC-ESI-MS with Sub-3-µm- ParticlesA. W. Perala, C. J. Toher, C. Marshall-Waggett, G. A. Valaskovic; New Objective, Inc., Woburn, MA, United States.

To enhance chromatographic resolution, analytical high-performance liquid chromatography (HPLC) has fol-lowed a distinct evolution toward smaller-diameter packing materials, at the expense of increased operating pressures. We herein investigate and compare the use of nanobore columns possessing integrally fritted tips packed with con-ventional (5- and 3.5-µm diameter) and smaller-diameter (2.5 to 1.7 µm) silica-based C18 media. Given operating pressure limits (~5000psi) of conventional HPLC, column theory predicts reduced-particle-size sorbents are feasible with shorter-bed columns. Direct on-column injection of standard peptide and protein digests subjected to tradi-tional reverse-phase, nanobore-pump-facilitated gradients reveal stable back pressure and high-caliber chromato-graphic data (6–12 sec peak width) throughout the analysis. Sub-2-µm particles packed to ~1–2-cm bed lengths display similar separation behavior, resolution, and reduced run times (≤50%) to that of 5-cm beds containing 5-µm par-



ticles. Employing sub-2-µm particles packed in longer bed (≥5 cm) lengths remains under investigation.

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Multidimensional Liquid Chromatography of Proteins Using Monolithic Weak Anion Exchange and Reversed-Phase ColumnsE. Sneekes1, R. van Ling1, B. de Haan1, B. Dolman1, R. Swart1, M. van Gils2; 1Dionex, Amsterdam, The Netherlands, 2Dionex Corp., Sunnyvale, CA, United States.

Recent developments in monolithic column technology have yielded significant improvements in the biopolymer analysis field. Monolithic columns offer several advantages over particulate columns due to their macroporous struc-ture, which provides fast mass transfer, low back pressure and high resolution. Polystyrene divinylbenzene (PS-DVB) monolithic columns are chemically inert; they offer high pH stability and excellent chromatographic performance in reversed-phase LC. While the application of reversed-phase monolithic columns is quite common, the use and avail-ability of functionalized monolithic columns is currently small. Thus, monolithic columns with anion exchange properties were developed, making fast 2D-LC analysis of complex protein samples possible. Simple fraction collec-tion between the two chromatographic dimensions yields even greater method flexibility. In addition, proteins are enriched into less complex fractions, which provides higher sequence coverage and improved confidence in identifica-tion. Proteins were separated on polymethacrylate-based monolithic weak anion exchanger and polystyrene divinyl-benzene-based reversed-phase columns. The orthogonal-ity, peak capacity, and robustness of the 2D-LC method are studied using a set of protein standards and a bacterial protein lysate.

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Fully Automated Off-Line Multidimensional LC Methods in ProteomicsR. van Ling1, B. Dolman1, E. Sneekes1, R. Swart1, M. van Gils2; 1Dionex, Amsterdam, The Netherlands, 2Dionex Corp., Sunnyvale, CA, United States.

Multidimensional liquid chromatography (MDLC) coupled to mass spectrometry is a valuable strategy for bottom-up or top-down workflows in proteomics. A large number of different MDLC approaches have been described for the separation of intact proteins and peptides.

Off-line MDLC techniques have several advantages over on-line approaches, including: (1) higher flexibility with respect to column dimensions and mobile phase selec-tion, (2) easier method development and, (3) the ability to re-analyze the fractionated effluent.

Here we present a fully automated method for off-line MDLC of peptides and proteins. The method allows for a combination of different column dimensions and chemis-tries to optimize the MDLC separation. Effluent from the first dimension column is directed to an eight-port injec-

tion valve and fractionated through the injection needle in a well plate or sample vials.

The second dimension separation utilizes a column-switching configuration for on-line desalting, sample con-centration, and direct interface to mass spectrometer. Alter-natively, for protein separations, a second fractionation step can be implemented after the reversed-phase separation to allow for proteolytic digestion.

Methods have been developed for 2D-LC of peptides and proteins using ion-exchange and reversed-phase chro-matography. Fractionation of the ion-exchange separation and subsequent injection of the fractions is performed automatically onto capillary PS-DVB monolithic columns. The fast mass transfer kinetics of the monolithic columns allows for fast gradient separations and a reduction of the total analysis time of the 2D-LC method. Interfacing the 2D-LC separation with tandem MS sequencing of peptides shows that peptides elute typically in one or two strong cation-exchange fractions. In addition, the system shows good precision performance for injection and fractionation of samples.

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Optimizing Separation Conditions for the Isolation and Purification of Synthetic PeptidesT. E. Wheat, J. Jablonski, E. L. Gildea, J. R. Mazzeo; Waters Corporation, Milford, MA, United States.

Peptides are used in basic research as well as in biophar-maceutical development. It is common to use synthetic pep-tides rather than isolating natural products. Such synthetic products may not be sufficiently homogeneous for use in experiments requiring bioactivity and specificity. Isolation and purification, therefore, become essential steps in a pep-tide-synthesis facility. We have examined the factors that influence purification protocols. This includes selection of a column, operating parameters based on the properties of the peptides, and the intended use of the product. A panel of synthetic peptides covering a range of properties includ-ing size, hydrophobicity, and isoelectric point is used for these evaluations. These test samples are used to measure the importance of pore size and particle size. Under these controlled conditions, there is little difference between 130 Å and 300 Å up to 40 residues. More resolution was observed with 5-µm particles than with 10 µ. The uses of the purified peptides may constrain the mobile-phase con-stituents. The suitability of biocompatible mobile phases, including acetic acid in place of trifluoroacetic acid and ethanol instead of acetonitrile affected the selectivity of the separation. In some comparisons, selectivity effects could be related to the peptide sequence, while other changes are still open to interpretation. An additional set of experiments has been performed investigating conditions for classes of peptides that have extreme or special properties. We show the impact of elevated temperature as well as the use of high pH and/or alternative solvents for peptides that are very difficult to dissolve in common chromatographic solvents. These experimental observations are combined into a sug-gested protocol for developing an isolation method.



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Nano-Scale Ion-Pairing Ultra-Performance Liquid Chromatography Coupled with QTof Mass Spectrometry for the Separation Analysis of PhosphopeptidesY. Yu, J. Ahn, M. Gilar, J. C. Gebler; Waters Corporation, Milford, MA, United States.

The identification of phosphorylated peptides by LC-MS is a challenging task. It is in part due to the relative low abundance of phosphopeptides; and their low ionization efficiency in positive ESI-MS ionization mode. The prob-lem is further exacerbated by the phosphopeptides’ ability to form complexes with metals such as Fe (III) or Al (III) accumulated in the LC systems. Thus, the amount of phos-phopeptides eluted from the LC system may be less than the amount injected, especially for multiply phosphorylated species. It has been reported that adding chelating agents such as EDTA into the sample before LC injection can dis-rupt the formation of phosphopeptide-metal complexes and improve the phosphopeptide detection. However, EDTA introduces several high background ions upon mass spec-trometry detection.

We utilized mobile phases with triethylammonium ion-pairing agent to eliminate the loss of phosphopeptides in a nano-scale ultra-performance liquid chromatorgraphy. Triethylammonium ion pairs with the phosphate group(s) in peptides and prevents the binding of phosphopeptides to metalic surfaces in LC systems. Direct comparison reveals a better recovery of phosphopeptides from nanoLC and LC systems with triethylammonium-based mobile phases (UV and MS detection). When using the same LC systems with conventional mobile-phase additives (0.1% formic acid), the majority of the multiply phosphorylated peptides were not detected. Triethylamine concentrations were optimized to obtain the desirable separation of phosphopeptides. The addition of EDTA to samples was no longer necessary to recover multiply phosphorylated peptides. ESI positive ion mode detected mostly singly charged phosphopeptide ions; extensive adduct ion formation (triethylamine/phos-phopeptide) was observed. Triethylamine tends to form adduct ions exclusively with phosphopeptides, which can be used as a diagnostic tool to identify phosphopeptides. These adduct ions were dissociated by Tee-in formic acid–containing solvent through an auxiliary pump before their entrance into the ESI source prior to the MS/MS (DDA) experiment.

identification of post-tr ansl ational modifications

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Phosphopeptide Enrichment Using a High-Affinity Metal Oxide Sorbent Packed in a Micro-Elution PlateJ. Ahn, Y. Yu, M. Gilar, A. Dubey, J. C. Gebler; Waters corporation, Miford, MA, United States.

The reversible phophorylation of serine, threonine, and tyrosine is one of the most important post-translational modifications involved in various cellular functions. Iden-tification of phophorylation sites by mass spectrometry is challenging due to the low abundance of phosphopep-tides and their limited ionization efficiency. Therefore, it is critical to selectively enrich the phosphopeptides prior to MS analysis. In this study we investigated the affinity extraction of phosphopeptides using a metal oxide–based solid-phase extraction (SPE) microscale device. This sor-bent shows high affinity towards phosphopeptides; acidic peptide adsorption is greatly minimized. When dealing with highly complex samples (e.g., cell lysate), the selectiv-ity of phosphopeptides can be further improved by mixing the sample with additive, aromatic carboxylic acids prior to loading onto the SPE. The metal oxide SPE is compared with other methods such as immobilized metal affinity chromatography and titanium dioxide–based phospho-peptide enrichment. Enhanced performance was observed in terms of the selectivity. MALDI-TOF and nanoLC/MS/MS were used to study the phosphopeptide recovery and selectivity. Phosphopeptide standards, alpha-casein tryptic digest, and yeast cell lysate were used to evaluate the phos-phopeptide enrichment method’s performance.

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Efficient Identification of Phosphorylation Sites in AMPK: Autoradiographical Visualization of Microfractionated Phosphopeptides using 384-PAC MALDI PlatesR. A. Brunisholz1, R. Tuerk2, D. Neumann2, Y. Auchli1, R. Schlapbach1, T. Wallimann2; 1Functional Genomics Center ETH, CH 8057 Zuerich, Switzerland, 2Institute of Cell Biology, ETH-Hoenggerberg, CH 8093 Zuerich, Switzerland.

Reversible phosphorylation of proteins plays an impor-tant role in many cellular processes. To investigate the functions of kinases and their substrates in vivo and in vitro—for example, production of phospho-specific anti-bodies or generation of phospho-site mutants—the precise identification of phosphorylation sites is mandatory.

A rapid and efficient workflow has been elaborated using prespotted 384 alpha-cyano-4-hydroxycinnamic acid MALDI targets as fractionation units; these are ide-ally suited to visualize 32P-labeled phosphopeptides. Ingel-tryptic digests of phosphorylated AMP-activated protein kinase (either by protein kinase B or autophosphorylation in the presence of γ32P-ATP) were separated on an Agilent



1100 capillary LC system coupled to a microfractionation unit. The tryptic peptides were deposited in 1-µL portions onto a PAC-MALDI target with 384 prespotted matrix preparations as well as calibrant spots. In order to visual-ize 32P-labeled peptides, the PAC plate was then exposed to a Kodak MR autoradiography film. Accordingly, the 32P-positive spots were screened for phosphorylation sites by performing MS and MSMS with the Ultraflex II TOF/TOF.

This new workflow for phosphorylation site identi-fication eliminates tedious manual handling of digested samples, thus reducing the initial sample material dramati-cally.

Furthermore, we observe that the separated peptides can be stored on the PAC plate for several weeks without any significant loss of resolution and signal intensity.

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The Insulin Granule: Probing Intracellular Post-Translational ProteolysisC. M. Buchanan, G. J. S. Cooper; University of Auckland, Auckland, New Zealand.

Here, we report the isolation and identification of a number of low-molecular-weight proteins (<10 kDa) from pancreatic β-cell granules. To date, most reported studies have employed two-dimensional electrophoresis to separate insulin granule proteins. However, standard 2DE is unable to resolve proteins of molecular mass <10 kDa, and we designed our current proteomic analysis to target the pool of small regulatory peptides contained within the β-cell granule. We used a single-step method to enrich granules for analysis by LC-MALDI and LC-MS, with subsequent protein sequencing.

Our results indicate the β-cell granule to be a rich source of peptides, obtained proteolytically from larger precursor molecules. We fully describe two novel peptides, both likely derived from intragranular proteolytic process-ing of pro-IGF-II.

It is commonly accepted that intracellular proteolysis occurs in neuroendocrine and endocrine cells, liberating physiologically active secretory peptides from larger pre-cursors. Processing begins co-translationally at the rough endoplasmic reticulum, where the signal peptide is removed from the prepro-hormone by a signal peptidase. The pro-hormone is then routed through the Golgi apparatus before being co-packaged with proteases into secretory vesicles destined either for the constitutive or regulatory pathway. The majority of the endoproteolytic processing is thought to occur in these secretory vesicles, with controlled prote-olysis at the C-terminal side of dibasic amino acids (usually RR or KR), as well as at selected mono-basic residues (both of which are also recognized by trypsin), and more spe-cific proteolytic motifs. Our methods allowed us to obtain information about the intact molecules (including proteo-lytic PTMs) that would otherwise be lost using proteomic methods that utilize tryptic digestion (e.g., MuDPIT or PMF).

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Phosphopeptide Enrichment and Analysis Using a Combination of Metal Oxide Affinity Purification and LC-MALDI MSE. Claude1, M. Snel1, Y. Yu2, J. C. Gebler2, T. McKenna1, J. I. Langridge1; 1Waters Corporation (MS Technologies Centre), Manchester, United Kingdom, 2Waters Corporation, Milford, MA, United States.

Phosphorylation is an important regulator of cell func-tion in eukaryotes. This post-translational modification can alter protein localization, regulate protein function, and stabilize and mediate their interactions. Due to their associ-ated negative charge, phosphopeptides are often poorly ion-ized compared to their non-phosphorylated counterparts, and their analysis is often complicated due to their low cel-lular abundance. Therefore, it is critical to selectively enrich the phosphopeptides prior to MS analysis.

In this study, we have evaluated a new way of enrich-ing phosphopeptides by using a metal oxide–based solid-phase extraction (SPE) microscale device where the eluent is analyzed by LC MALDI MS. This sorbent has a high affinity for phosphopeptides, and the problem of acidic peptide adsorption associated with immobilized metal-ion affinity chromatography, the previous technique, is greatly minimized.

Furthermore, recent developments in MALDI and LC-MALDI spotting devices allow the coupling of the off-line chromatographic separation step to the subsequent MS analysis.

Data will be presented showing the enrichment of phos-phopeptides from a digest of a single phosphoprotein, beta casein, analyzed by LC-MALDI MS, where enhanced per-formance was observed in terms of the selectivity of the phosphopeptides. Further analysis of more complex pro-tein mixtures (six and twelve standard proteins) which contain phosphopeptides will be presented, demonstrating the selectivity achieved using an aromatic carboxylic acid additive.

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Global Analysis of Phosphotyrosine Sites in Src-Transformed Cells: Improved Methods for Sample Preparation and AnalysisA. L. Ham1, S. Hill1, S. K. Hanks2; 1Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN, United States, 2Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, United States.

Interest in tyrosine phosphorylation patterns has been sparked since increases in phosphotyrosine have been asso-ciated with malignancy and tumor formation. We have employed several methods to investigate the phosphoty-rosine profile of Src-transformed cells, including an exten-sive study using the methods published by Rush et al.1 In order to streamline the Rush et al. protocol, we have gener-ated peptides by running the lysate approximately 1.5 cm into a 10% bis-tris polyacrylamide gel (with reduction and alkylation prior to the gel) followed by in-gel digestion.



Phosphotyrosine-containing peptides from tryptic diges-tions were enriched using either the pY100 antibody or the 4G10 antibody. Phosphotyrosine-containing peptides were eluted off the beads as described by the Rush et al. protocol or with the addition of an acetonitrile elution of the beads. Samples were analyzed on a Thermo LTQ by reverse-phase chromatography using data-dependent analy-sis. The results were searched using the Sequest algorithm and the mouse subset of the Uniref100 database with con-catenated reverse database searching for false-positive esti-mation. Results were filtered in a two-step process based on multiple Sequest scores, with increased stringency for novel sites. Results from multiple biological replicates were compared with those that we obtained using the Rush et al. protocol, and they produced comparable results. Pep-tides accepted from multiple replicates resulted in a false-positive rate of less than 3%. The addition of an acetoni-trile wash increased the elution of peptides from the beads and increased identifications. This procedure saved 1–2 d for sample preparation with equivalent or improved results. We are currently exploring digestion of whole-cell lysates in trifluoroethanol to further streamline this protocol, and the use of the LTQ-Orbitrap to more confidently identify the sites.

reference

1. Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotech 2004;23:94-101.

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Despite Rumors to the Contrary, PCNA Really IS PhosphorylatedD. H. Hawke, S. Wang, Y. Nakajima, M. Hung, R. Kobayashi; UT-M.D.Anderson Cancer Center, Houston, TX, United States.

Proliferating cell nuclear antigen (PCNA) is a small nuclear protein that plays a central role in DNA replica-tion. Although it has been intensely studied, there are strangely few reports of post-translational modifications to this protein. A site of ubiquitination has been described, and some have claimed the protein is not phosphorylated. A recent publication proposed partial methyl esterification of a number of Glu and Asp residues. Very recently, we found a phosphorylated Tyr (211) and showed it regulates stability of the protein. Increased phosphorylation at this site coincides with pronounced cell proliferation, and also correlates better with poor survival of breast cancer patients than total PCNA level. We describe here the strategy and experimental details of how we found Tyr211 to be phos-phorylated.

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Identification of Glycosylation Sites Using Electron Transfer Dissociation in a Linear Ion TrapY. Huang1, Z. Hao1, S. Twine2, J. Mullen2, K. Waddell1, J. Kelly2; 1Thermo Fisher Scientific, San Jose, CA, United States, 2Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada.

Glycosylation of proteins plays an important role in bio-logical systems. The attachment of polysaccharide chains serves various key cellular functions. Thus, it is often criti-cal to determine the exact site of glycosylation. Tandem mass spectrometry (MS/MS) employing linear ion trap technology has emerged as a cornerstone for protein identi-fication and PTM analysis in bottom-up proteomics. How-ever, the glycosylation moiety is usually fragile and falls off easily during the collision-induced dissociation (CID) MS/MS activation process without leaving much evidence of its sites of attachment. Electron transfer dissociation (ETD) has recently been commercially introduced onto a linear ion trap. This new fragmentation methodology shows great promise in its ability to preserve the saccharide side chains on a peptide, while obtaining fragmentation between the α-carbon and the nitrogen throughout the backbone. This enables the exact glycosylation site to be deduced from the MS/MS spectrum.

N-linked glycopeptides from bovine fetuin and O-linked glycopeptides from a glycoprotein isolated from a Gram-positive bacterium were analyzed on a Finnigan LTQ with the added ETD functionality. While the O-linked glycans have residue masses around 400 Da, the N-linked glycan is well over 2800 Da. ETD MS/MS spectra showed contiguous c and z peptide fragment ion series preserving the sugar intact for N-linked peptides as well as O-linked peptides. The sites of the O-linked glycosylation deduced from the ETD MS/MS spectra have been confirmed with chemical degradation (beta-elimination) studies.

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Using Titanium Dioxide IMAC for Enrichment of Phosphopeptides Prior to Tandem Mass SpectrometryJ. Zhu, Q. Lin; University at Albany, Rensselaer, NY, United States.

Protein phosphorylation plays a significant role in regu-lating cellular processes such as signal transduction, cell division, cell motility, apoptosis, metabolism, differentia-tion, gene regulation, and carcinogenesis. Typically, there are 10–20% of proteins which are phosphorylated. Due to the low level of phosphoproteins in the presence of over-whelming amounts of non-phosphorylated proteins, as well as those proteins’ wide dynamic variation over time, identification of phosphopeptides is still a formidable task. In addition, phosphopeptides often have poor ionization efficiency in MS analysis. Thus, a highly sensitive detec-tion method plus phosphopeptide enrichment is extremely important for a successful phosphopeptide identification. Currently, immobilized metal affinity chromatography (IMAC) is the method of choice for enriching phospho-peptides from complex biological samples. Typically, nickel,



iron, and gallium–based IMAC shows significant binding of non-phosphorylated peptides that have multiple acidic residues. Forest White et al. used a kind of chemistry to put methyl esters onto those acidic groups (D and E) to solve the problem of nonspecific binding to the IMAC beads. However, this approach brings in a lot of side reactions to that chemistry, and raises issues of how complete the modifications are. Recently, several papers and posters have been published demonstrating the unique ability of tita-nium dioxide and zirconium dioxide to selectively retain phosphopeptides contained in complex biological mixtures. In this application, a TiO2-based IMAC method was suc-cessfully developed to enrich phosphopeptides and adapted to a complex biological sample, Saccharomyces. Trapping phosphopeptides are demonstrated via the analysis protein CaO19_4593 (gi|68466366), a family of GTPase-activating proteins which contains multiple kinase-binding domains.

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High-Resolution Mass Spectrometry Study of the Effects of Chemical and Environmental Stimuli on the Post-Translational State of Histone H4L. Mackay1, S. Weidt1, T. Hupp2, P. Langridge-Smith1, L. Hayward2; 1University of Edinburgh, Edinburgh, United Kingdom, 2Edinburgh Cancer Research Centre, Edinburgh, United Kingdom.

As altered epigenetic regulation is a common feature of cancer, a greater understanding is required of the role of histone post-translational modifications as epigenetic regulators of changes in chromatin structure. Chromatin remodeling may also be important in the cellular response to DNA damage induced by anti-cancer drugs. A greater understanding of the nature of histone PTMs and result-ing DNA damage in normal and cancerous tissues might indicate differences in the activity of enzymes involved in modifying histones, which could yield potential targets for cancer-specific therapy. Trichostatin A (TSA), a histone deacetylase inhibitor, promotes acetylation of histones and subsequently increases the accessibility to cellular machin-ery by the unraveling of DNA.

Using high-resolution 9.4T and 12T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers, the aim of this work was to characterise, in an unbiased fashion, the histone PTMs induced in histone H4 in response to treatment with TSA and certain environmental stimuli.

FT-ICR mass spectrometry is not well suited to on-line analysis, owing to the extended scan time required to achieve optimum resolving power. The TriVersa Nanomate enables fraction collection of the HPLC eluent allowing FT acquisition time to be decoupled from separation time. Fol-lowing further sample cleanup, it proved possible to infuse unfractionated complex histone samples. The most abun-dant classes of histone present in the cell (H2A, H2B, H3, H4) were observed

The resolving power of FT-ICR permits the post-translational modification of H4 to be observed directly. It can be seen that in the case of cells treated with TSA, there was considerable variation in the acetylation state of histone H4. It was also possible to affect the acetylation state using certain environmental stimuli.

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Identification and Site Determination of PTM in FMIP Peptides Using Electron Capture Dissociation on a FTICR Mass SpectrometerW. Metelmann-Strupat1, J. R. Griffiths2, B. Delanghe1; 1Thermo Fisher Scientific, Bremen, Germany, 2Stem Cell and Leukaemia Proteomics Laboratory, Division of Cancer Studies, University of Manchester, Christie Hospital, Manchester, United Kingdom.

The Fms-interacting protein (FMIP, MW 78 kDa) acts as a shuttling protein during macrophage differentiation in the human immune system.1

Previous work on an enzymatic digest of this protein has revealed a peptide with a precursor mass corresponding to the FMIP peptide YTCQELQR with the addition of 80 Da. This mass difference may be attributable to either phos-phorylation or sulfonation. The type of post-translational modification (PTM) could not be determined by detailed analysis of the resulting MS/MS spectrum obtained under typical collision-induced dissociation conditions.

In order to distinguish the PTM, we analyzed the sam-ple on a Thermo Fisher LTQ-FT Ultra mass spectrometer making use of its very high and reliable mass accuracy. In addition, electron capture dissociation (ECD) was per-formed on the peptide in an attempt to assign unambigu-ously the site of modification.

The sample was separated using a nanoLC setup (Ther-moFisher Scientific Micro Autosampler and Surveyor MS Plus pump) equipped with a C18 trapping column and a C18 100 × 0.075 mm analytical column (both: www.nanoseparations.com). Standard HPLC solvents (MS grade) were used for the analysis: water/acetonitrile (98:2 v/v, 0.1% formic acid) as solvent A and acetonitrile/water (80:20 v/v, 0.1% formic acid) as solvent B.

The applied method used a FTMS full scan and three data-dependent ECD scans per cycle, picking the top three signals from the full scan and subsequently excluding these for 20 sec.

The analysis clearly reveals the type of modification as well as the site. The poster will present the data as well as their interpretation.

reference

1. Tamura T, Mancini A, Joos H, Koch A, Hakim C, Dumanski J. FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation. Oncogene 1999;18(47):6488–95.

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Selective Detection of Phosphoproteins and Subsequent Enrichment of Constituent Phosphopeptides for Phosphorylation Site Analysis by Mass SpectrometryA. Mikulskis, S. E. Kramer, A. Bogdanova, H. Xie, E. Golenko, Y. Wang, H. Lisoukov, W. F. Patton; PerkinElmer Life and Analytical Sciences, Waltham, MA, United States.

Protein phosphorylation is crucial to the global regula-tion of the intricate biochemical networks of cell signaling



pathways, allowing protein functional activity, stability, association into multi-subunit complexes, and subcellular localization to be integrated and tuned for generation of highly specific biological responses crucial to fundamental cellular functions. An antibody-free process workflow is described involving orthogonal phosphomonoester selec-tive binding strategies. First, complex protein samples, such as rat liver cytosol proteins, are separated by conventional gel electrophoresis and a fluorophore-conjugated alkoxide-bridged dinuclear zinc complex is employed to selectively highlight phosphoproteins via binding to the phospho-monoester dianion moieties of serine, threonine, and tyro-sine residues at neutral pH. Interaction with other anionic residues, including carboxylate residues on proteins, is insignificant. As little as 1 ng of phosphoprotein is detect-able by this method using standard charge-coupled device camera- or laser-scanner-based imaging systems. Then, phosphoprotein bands are excised and subjected to pro-teolytic digestion. Constituent phosphopeptides are subse-quently purified using titanium dioxide thin-film-coated magnetic beads at acidic pH. Phosphopeptides are eluted at alkaline pH and directly characterized by MALDI-TOF or tandem mass spectrometry, without chemical modifica-tion by methyl esterification. Phosphopeptides can readily be identified from as little as 78 fmol of starting material, with minimal contamination of samples by acidic peptides, as is often encountered using conventional trivalent ferric- or gallium-based metal ion affinity approaches. The high affinity and capacity titanium dioxide-based purification method is also suitable for the direct enrichment of phos-phopeptides from human serum, which could lead to new approaches for biomarker discovery from biological fluids.

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Targeted Mass Spectrometric Strategies for Global Mapping of Ubiquitination on ProteinsS. Mollah1, J. Lill2, D. Arnott2; 1Applied Biosystems, Foster City, CA, United States, 2Genentech,Microchemistry and Proteomics Laboratory, South San Francisco, CA, United States.

Protein modification by ubiquitin is an important event that represents a highly versatile means for regulation of protein function. Ubiquitination acts at multiple levels to regulate gene expression, including targeting for deg-radation, proteolytic activation, and intracellular localiza-tion. Mapping of protein ubiquitination sites is important because it can facilitate the elucidation of mechanisms of this modification, yielding insights into cell regulation. Recently, mass spectrometry has been employed for deter-mination of ubiquitination sites; however, this protein anal-ysis approach is incomplete and not saturated for any given protein. Existing targeted mass spectrometric approaches can monitor only a few predicted ubiquitinated peptides at any given time, thus requiring multiple analysis and samples. Additionally, proteins are typically enzymatically digested with trypsin. However, ubiquitinated peptides are modified on their lysine residues with the addition of GG motifs, thus generating large tryptic peptides that are often multiply charged. Hence, MS/MS spectra can be com-plicated and challenging to interpret. Here we employ a

unique approach to target and sequence sites of ubiquitina-tion on proteins and their relevant peptides.

Ubiquitinated proteins were purified and digested with various enzymes to obtain varying lengths of peptides. Sample analysis was performed using reverse-phase nano-HPLC interfaced to a hybrid quadrupole linear ion trap mass spectrometer. MRM-initiated detection and sequenc-ing workflow was used to monitor 70–100 predicted ubiq-uitinated peptide masses and their fragment ions in one analysis for simultaneous detection, identification, and relative quantification of the peptides. A number of ubiq-uitinated peptides were identified by this analysis, and the results will be discussed. This targeted approach has been shown to provide a high-throughput analysis in addition to better specificity and sensitivity for ubiquitination site mapping.

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Automated Analysis of Gel-Derived Phosphoproteins Using the Investigator Proteomic SystemD. A. Moraga1, M. C. Chow1, I. Isaac2, S. M. Stevens, Jr.1; 1University of Florida, Gainesville, FL, United States, 2Genomics Solutions, Ann Arbor, MI, United States.

The evolution of proteomics-based technologies has led to the development of powerful analytical methods for the analysis of post-translational modifications such as phos-phorylation. Chemical derivatization strategies that involve beta-elimination and Michael addition chemistries as well as chromatographic enrichment approaches using immo-bilized metal affinity chromatography have been success-fully applied to global phosphoproteome analysis. Recently, enrichment of phosphorylated peptides using TiO2 prior to mass spectrometric analysis has been shown to be a selective and robust method for phosphoprotein charac-terization. We present an automated workflow using TiO2 microcolumns for the enrichment of phosphorylated pep-tides derived from in-gel digested proteins in combination with the Investigator Proteomic System (Genomics Solu-tions, Ann Arbor, MI).

A pool of beta casein and ovalbumin (10 µg each) was used as a standard to test the automated phosphoprotein analysis workflow. This same workflow was then success-fully applied to characterize in vitro and in vivo phos-phorylation events of selected 14-3-3 proteins derived from Arabidopsis thaliana. Samples were separated by either 1D or 2D SDS-PAGE, stained with Pro-Q Diamond (phos-phoprotein-specific) fluorescent stain, and imaged using either a Typhoon 9400 scanner or the Investigator ProPic (Genomic Solutions). Putative phosphoprotein-containing spots were then excised and digested in-gel with trypsin using the Investigator ProPic and ProGest, respectively. Tryptic digests from each protein spot were processed with the ProMS workstation using TiO2 microcolumns (Gly-gen Corp., Columbia, MD) for phosphopeptide enrich-ment prior to MALDI-TOF/TOF analysis (ABI 4700 Pro-teomics Analyzer).



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Solid-Phase Strategy for Phosphorylation/Glycosylation Site MappingH. Nika, R. Hogue Angeletti; Albert Einstein College of Medicine, Bronx, NY, United States.

Chemically induced β-elimination of phosphate from serine and threonine coupled with Michael addition has emerged as a chemical strategy to address both the ion suppression and the gas-phase lability of the phosphate group. In previous work, we have adopted the chemistry to solid-phase derivatization on C18 ZipTip pipette tips using barium hydroxide as elimination base and 2-amino-ethanethiol as nucleophile.1 The utility of the protocol for improved phosphopeptide detection by signal enhancement was demonstrated with low-level amounts of tryptic pro-tein digests, and the resultant increased MS/MS spectral information content greatly facilitated mapping of the site of phosphorylation.

In this report, we have focused on chemistry optimi-zation of O-phospho and O-GlcNAc modified peptides reported as resistant to β-elimination, i.e., those containing the modified residues followed by proline. Conclusive map-ping of these phosphorylation sites has become increasingly important in view of the fact that phosphorylated Ser/Thr-Pro motifs are substrates for prolyl cis/trans isomerase Pin1.2 Similarly, unambiguous site determination of O-GlcNAc modifications has more recently attracted considerable interest because of the global and often site-specific recip-rocal relationship between O-GlcNAc and O-phosphate in many cellular responses.3 We have used a panel of model peptides to define the optimal reaction conditions for both concurrent and consecutive elimination/Michael addition reactions and employed carbon/C18 mixed-phase ZipTips to afford efficient binding of small hydrophilic peptides.

references

1. Nika H, Hawke DH, Kobayashi R. Derivatization on reversed-phase support for enhanced detection of phosphorylated pep-tides. 51th ASMS 2003, Montreal, ThPN 282.

2. Ryo A, Liou Y-C, Lu KP, Wulf G. Prolyl isomerase Pin1: A catalyst for oncogenesis and a potential therapeutic target in cancer. J Cell Sci 2003;116:773-783.

3. Comer FI, Hart, GW. O-Glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phos-phate. J Bio Chem 2000;275:29,179–29,182.

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Multiplex Detection of Non-Phosphorylated and Ser 9–Phosphorylated GSK3β Isoforms Using 2D Gel Electrophoresis and a Fluorescent-Based Western Blotting SystemH. Nordvarg, S. Edlund; GE Healthcare Bio-Sciences AB, Uppsala, Sweden.

Western blotting systems are most commonly used after separation of proteins on one-dimensional (1D) SDS-PAGE gels. The fluorescent-based ECL Plex West-ern Blotting Detection System (GE Healthcare, Piscataway, NJ) provides sensitivity, linearity, and a dynamic range of

nearly four orders of magnitude, as well as the possibil-ity of multiplex analysis-detection of two protein epitopes on the same membrane simultaneously. Multiplexing is commonly used for quantitation of one protein relative to a protein of known abundance (housekeeping protein). It can also be used for the detection and quantitation of post-translational modifications such as phosphorylation, provided that there are antibodies available. In this study, a two-dimensional Western blotting approach was evaluated in addition to the traditional 1D workflow, using fluores-cent secondary antibodies, namely the ECL Plex Western Blotting Detection System. Human PC-3U cell lysate was separated by 1D and 2D SDS-PAGE, followed by blotting and incubation with primary antibodies against non-phos-phorylated and Ser 9-phosphorylated forms of glycogen synthase kinase-3 beta (GSK3β). ECL Plex CyDye Cy3- and Cy5-conjugated secondary antibodies were used for simultaneous detection of the non-phosphorylated and phosphorylated forms of GSK3β. The 1D Western blot-ting experiment showed that both the non-phosphorylated and phosphorylated forms of GSK3β could be detected in PC-3U cells without transforming growth factor beta (TGF-β) activation. The 2D Western blotting experiment gave information on phosphorylation states that could not be obtained by 1D Western blotting. A 48-kDa band corre-sponding to phosphorylated GSK3β in the 1D experiments was resolved into at least five distinct protein isoforms, two of which were phosphorylated at serine 9.

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Automated Analysis of Phosphopeptides by Immobilized Metal Affinity ChromatographyS. Rao, P. McCarthy, M. L. Tracy, N. Avdalovic, C. Pohl; Dionex Corp., Sunnyvale, CA, United States.

Protein phophorylation is a widespread post-translational modification that plays a crucial role in many cellular processes. However the analysis of phosphopep-tides and reproducibility of methods is not easy because of many factors, among which relatively low abundance makes it a challenging analytical process.

We present an automated method for the phophopep-tide analysis using an immobilized metal affinity chro-matography (IMAC) column to enrich phosphopeptides and subsequent reverse-phase HPLC analysis. The recently developed IMAC is an analytical HPLC column that can be used repeatedly and reproducibly. By using a dual pump and automatic sample injections, the entire phophopeptide analysis is automated and is completed in about an hour.

We used tryptic digestions of beta-casein as the model to evaluate nonspecific binding of some nonphosphorylated and all phosphorylated peptides to the IMAC column under the loading conditions (20 mmol/L formic acid). Under the release conditions at pH 9 with ammonium formate, all the expected phosphopeptides are released and recovered quantitatively. The data are presented to show the specific-ity, recovery, and reproducibility of the method.



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Antibody Arrays for Measuring Site-Specific Protein PhosphorylationC. Rauh-Adelmann, N. F. Gordon, J. R. Graham, C. E. Murphy, J. I. Boucher, L. G. Yen, T. K. Nadler; Epitome Biosystems, Waltham, MA, United States.

We have developed a sandwich immunoassay method for quantitatively measuring site-specific phosphorylation of multiple proteins and at multiple sites within a protein simultaneously, without the need for multiple, phospho-site-specific detection antibodies. This approach utilizes antibodies to capture a unique peptide sequence (EpiTag) proximal to the phosphorylation site of interest. The cap-tured peptides containing the phosphorylation site(s) of interest are then interrogated with a commercially avail-able, pan-anti-phospho antibody to determine whether the site is indeed phosphorylated. Unlike traditional immuno-assays, select epitopes are made available to the antibodies by a unique sample-processing procedure. The procedure involves proteolytic fragmentation of proteins in order to segregate multiple phosphorylation sites within a given pro-tein. Since proteolytic cleavage is predictable, quantitation is achieved by interpolating protein concentrations from standard curves generated with synthetic phosphopeptide standards. This approach obviates the need for phospho-proteins, which can be difficult to generate and qualify. The sandwich immunoassays demonstrate titration over a large dynamic range (≥3 logs) and achieve sensitivity at or below picomolar concentrations of standard. In contrast, no sandwich formation is observed with standards that have been dephosphorylated with lambda phosphatase. While the approach is limited to measuring previously discovered phosphorylation sites and does not distinguish between multiple sites that lie within the same proteolytic fragment, this proprietary approach is directly applicable to both bead-based and planar arrays, and has been multiplexed and used to measure >30 phosphotyrosine sites on vari-ous targets across multiple signaling pathways. In addition, the approach is currently being evaluated for multiplexed measurements of other phosphoresidues, and we believe this technology can be applied specifically, to study and quantify particular signaling events, as well as on a more global level, to identify and “fingerprint” activated signal-ing pathways during different disease states or in response to therapeutic strategies.

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MALDI-MS Analysis of Phosphopeptides Complexed with Metal IonsJ. D. Read, W. H. Fischer; The Salk Institute, La Jolla, CA, United States.

Objective: To develop a procedure that can serve to identify phosphopeptides in complex mixtures.

The identification and characterization of phospho-peptides by mass spectrometry is an active area of investiga-tion due to the importance of the modification in a variety of regulatory processes. Phosphopeptides exhibit high bind-ing affinity for a number of metal ions, including iron(III) and gallium(III). We have investigated whether MALDI-

MS allows the detection of cationized phosphopeptides. A number of metal ions, including Fe(III), Ga(III) and Ca(II), were tested in a variety of matrices. It was found that Fe(III) binds preferentially to phosphopeptides in the presence of non-phosphorylated peptides under acidic conditions.

Preliminary results: When incubating phosphopep-tides with millimolar concentrations of metal salts (FeCl3, Ga(NO3)3, CaCl2), cationized species were observed in MALDI-MS spectra with all matrices tested. The major cationized species carried a single positive charge, e.g., [M –2H +Fe]+. The cationized species typically exhib-ited a signal intensity 10–20% that of the protonated one ([M+H]+). To test the specificity of the metal ion binding, a model phosphopeptide (beta-casein 48-63) was mixed with a BSA tryptic digest and incubated with metal salts on the MALDI target in the presence of matrix. Cationization was observed to a varying extent in all cases, but only one metal ion/matrix combination exhibited specificity for the phosphopeptide. Iron(III) bound to the phosphopeptide exclusively when employing ACHC as the matrix. A vari-ety of phosphopeptides containing phosphorylated serine, threonine, and tyrosine residues were analyzed in the same manner with similar results. We were able to obtain TOF/TOF fragmentation spectra when selecting the [M+H]+ ion corresponding with the phosphopeptides but not for the cationized precursor. Currently, conditions are being tested that increase the specificity of Fe(III) for phosphopeptides as opposed to other acidic and potentially chelating pep-tides.

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Characterization of Pil1p and Lsp1p Phosphorylation in S. cerevisiae using Tandem Affinity Purification and LC-MS/MSL. M. Regalla, S. Stevens, Jr., T. J. Lyons; University of Florida, Gainesville, FL, United States.

In order to elucidate the signaling pathway of the human mPR-α protein, a membrane progesterone recep-tor, a strain of S. cerevisiae overexpressing this protein was generated. Initial global screens using 2D gels, a phospho-specific stain, and tandem MS/MS identified 35 proteins exhibiting alterations in phosphorylation state in response to progesterone activation. Among these were Pil1p and Lsp1p, components of a newly discovered immobile pro-tein mass, the eisosome, purported to denote endocytotic sites. However, due to their similar molecular weights, pI values, and a shared sequence identity of 72%, the separa-tion achieved using 2D gel electrophoresis was insufficient to identify which of these two proteins was exhibiting a change in its phosphorylation state. The experiment pre-sented here utilizes a strain of yeast containing an endog-enously TAP (tandem affinity purification)-tagged version of Lsp1p. Since Pil1p and Lsp1p interact, the purification pulled out both of these eisosome proteins. Subsequent sep-aration using 1D gels distinguished the two proteins due to the additional molecular mass imparted to Lsp1p by the tag. Both proteins were then excised, digested in-gel with trypsin, and analyzed via LC-MS/MS using a Q-Star XL mass spectrometer. A total of six unique phosphopeptides were identified: five in Pil1p and one in Lsp1p. The tag was



then swapped onto Pil1p in order to identify any additional sites on Lsp1p that may have been suppressed due to the presence of the tag. The findings presented here represent a significant step towards our ultimate goal of monitoring changes in phosphoproteins in response to progesterone activation.

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Rapid and Individual-Specific Glycoprofiling of the Low-Abundant Plasma Protein Tissue Inhibitor of Metalloproteinases-1M. Thaysen-Andersen1, I. Thøgersen2, U. Lademann3, N. Brünner3, J. Enghild2, P. Højrup1; 1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 2Department of Molecular Biology, University of Aarhus, Aarhus, Denmark, 3Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.

A gel-based method for a mass spectrometric (MS) site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydro-philic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glyco-profiling was performed using matrix-assisted laser desorp-tion/ionization MS and MS/MS. The method proved to be fast and sensitive, and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant pro-tein, both comprising N-glycans of a highly heterogeneous nature.

To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low-abundant (50–80 ng/mL) N-glycosylated plasma protein was purified in an individual-specific manner from five healthy indi-viduals using IgG depletion and immuno-affinity chroma-tography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and tri-antennary complex oligosaccharides. Additionally, it was shown that platelet-derived TIMP-1 displayed a similar glycoprofile. This is the first study to investigate the gly-cosylation of naturally occurring human TIMP-1 and the high similarity of the glycoprofiles showed that individual-specific glycosylation variations of TIMP-1 are minimal. In addition, the results showed that TIMP-1 derived from platelets and plasma is similarly glycosylated. This compre-hensive and rapid glycoprofiling of a low-abundant glyco-protein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus lead to early detection and monitoring of diseases.

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Monitoring of Temporal Changes in Phosphorylation States of Proteins Using Mass Spectrometry and a Chemical Labeling StrategyF. Tsuchiya1, M. Matsubara2; 1Applied Biosystems Japan, Tokyo, Japan, 2Kyoto Gakuen University, Kyoto, Japan.

Introduction: Protein phosphorylation is one of the most important post-translational modifications that regulate the function, localization, and binding specificity of target proteins. Specifically, protein phosphorylation works as a switch to turn “on” and “off” protein activity or a cellular pathway. Protein phosphorylation is also known to be both spatial and dynamic in its distribution. Therefore, a work-flow that provides the detailed information of phosphoryla-tion site location, site occupancy under specific conditions, etc., is necessary. Current strategies such as ELISA, radio isotope workflow, and Western blotting lack the ability to ascertain independent information of each phosphorylation site. Our mass spectrometry–based method using a chemi-cal labeling strategy provides dynamic information about phosphorylation on each phosphorylation site.

Method: A model system using JNK1 kinase and the substrate protein myelin basic protein (MBP) was selected. JNK1 phosphorylates serine and threonine residues just before the proline residue, typical of this MAP kinase fam-ily. The MBP substrate was incubated with JNK1 in vitro, and samples were removed at four different time points (0, 5, 30, and 60 min). These samples were digested and labeled with iTRAQ reagent in accordance with the stan-dard procedure. A nano-LC-MS/MS experiment was per-formed using the 4000 Q TRAP System, and the data were analyzed by ProteinPilot Software.

Results: All of the peptides possessing a possible phos-phorylation site motif in MBP were detected. For each identified peptide, the iTRAQ reagent reporter ions clearly showed the change in abundance of specific MBP pep-tides as they become phosphorylated by JNK1 over the time course of the assay. These results indicate that this workflow is a robust strategy for routinely characterizing the variation of phosphorylation through the interaction between protein substrate and kinase.

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Fractionation or Affinity Purification? Using Microcolumns (TiO2, SCX, HILIC) to Isolate PhosphopeptidesA. J. Ytterberg, R. R. Ogorzalek Loo, P. Boontheung, J. A. Loo; UCLA, Los Angeles, CA, United States.

Phosphorylation is one of the most common of the reversible post-translational modifications, but also one of the most challenging. During the last few years, phospho-peptide analysis has benefited from the rapid improvement in biological mass spectrometry, but also from innovations such as immobilized metal affinity chromatography, the development of antibodies specific for phosphorylated amino acids, and most recently, chromatography using TiO2.

During our analysis of the saliva phosphoproteome, we were concerned that there is a bias against monophosphor-ylated peptides when using TiO2 columns. We therefore


�0 JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007

decided to explore alternatives that rely on fractionation rather than affinity purification. Using the approach of Roepstorff and colleagues of packing columns in pipette tips to make disposable microcolumns, and combining strong cationic exchange (SCX) together with hydrophilic interaction liquid chromatography (HILIC), we have been able to fractionate peptides from digests of total saliva, and isolate phosphopeptides in a few fractions with a minimum of non-phosphorylated peptides. In preliminary experi-ments using about 6 µg of saliva, we have been able to map out phosphorylation sites from proteins such as the salivary acid proline-rich phosphoprotein 1/2, the salivary α-amy-lase, and statherin. A comparison of isolation of phospho-peptides using TiO2 vs. a combination of SCX and HILIC will be presented.

macromolecul ar inter actions

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A Demonstration of the Application of Ion Mobility Mass Spectrometry for the Analysis of Activated BiomoleculesT. McKenna, I. Campuzano, K. Giles, J. I. Langridge; Waters Corporation (MS Technologies Centre), Manchester, United Kingdom.

Electrospray ionization (ESI) is a very gentle form of ionization, which enables the intact transfer into the gas phase and detection of large multi-protein structures with little or no fragmentation; when ESI is coupled to mass spectrometry, non-covalently assembled macromolecular protein complexes can be detected and accurately mass measured.

The transfer of non-covalently associated protein-protein complexes from solution to the gas phase generally results in the formation of ions possessing relatively few charges; the m/z values are often above 10,000. In some of the data presented in this poster, MS/MS activation of such biomolecular complexes can produce ions with m/z values in excess of 20,000.

By coupling an ion mobility separator (IMS) with a time-of-flight (ToF) mass spectrometer, one can not only accurately mass measure intact biomolecular complexes, but also one can measure their collisional cross-sections and differences in cross-section produced upon activation, and detect subtle conformation differences, which are not evident from spectral data alone.

Here, we show the analysis of several different, non-covalently associated protein-protein complexes, which differ vastly in mass and collisional cross-section, by IMS-ToF-MS and IMS-ToF-MS/MS.

P88-S

Macromolecular Interactions: PPARy Ligands Enhance Phagocytic Clearance of Apoptotic MacrophagesK. A. Gilford1, E. Thorp2, I. Tabas2; 1Brooklyn College, Brooklyn, NY, United States, 2Columbia University, New York, NY, United States.

Peroxisome proliferator-activated receptor (PPAR)γ ligands decrease early atherosclerosis lesions in experimental animal models, but their effects on advanced atherosclero-sis are not known. A unique process in advanced atheroscle-rosis is the accumulation of free cholesterol (FC) by macro-phages (Mfs), which, according to previous in-vivo studies, can result in Mf death, lesional necrosis, inflammation, and plaque susceptibility to rupture. Mf apoptosis occurs through all stages of atherosclerosis, and inefficient phago-cytic clearance of dying Mfs may lead to cellular necrosis and release of plaque-destabilizing factors. Here we sought to determine how PPARy agonists would affect phagocyto-sis of FC-induced apoptotic cells. We found that with the use of these PPARγ agonists, phagocytosis of these apop-totic cells increased significantly. This enhanced phago-cytic uptake was associated with increased accumulation of filamentous actin at the interface between phagocyte and apoptotic cell, suggesting an effect of PPARy ligands on intracellular actin signaling. Indeed, macrophages treated with the PPARy ligand rosiglitazone had decreased levels of the actin-modulating protein RhoA. Thus, compounds designed to activate PPARy, inducing those in clinical use such as rosiglitazone and pioglitazone may reduce athero-sclerotic maturation by enhancing phagocytic clearance of dying macrophages. Future derivations of PPARγ ligands may be selected to further exploit this phagocytic prop-erty.

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Protein Complex Analysis by Chemical Cross-Linking and High-Mass MALDI Mass SpectrometryA. Nazabal, R. Wenzel; CovalX, Zürich, Switzerland.

The development of new methodology for the direct analysis of protein-protein interactions is of high interest, as protein complexes are involved in all cellular processes. If mass spectrometry is routinely used for protein analy-sis, it is still challenging to use this analytical tool for the analysis of protein-protein interactions. Most of the succes-ful studies in this field have been done using electrospray ionization, requiring time-consuming optimizations both on sample preparation and instrumental settings.

MALDI mass spectrometry is a faster method for the analysis of proteins but is rarely used for the direct analysis of protein-protein interactions. The principal reason is the high tendency of non-covalent interactions to dissociate during the ionization step. Another issue is the detection of the intact complexes as single-charge pseudomolecular ions.

Here, we present the analysis of intact protein com-plexes by MALDI mass spectrometry using the combina-tion of chemical cross-linking and high-mass MALDI mass


JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007 �1

spectrometry. To circumvent the dissociation problem, a specific chemical cross-linking reaction is performed prior to the MS analysis, maintaining the integrity of the intact complex. To allow the detection of the complex, a new high-mass detection system is used, allowing sensitive analysis (nM range) in the 10–1200 kDa range.

Different applications will be presented:Immunochemistry: Direct analysis of immuno-complexes

between prion/antiprion; HAtag/antiHA; GSTtag/antiGST; BSA/AntiBSA in the 150–400 kDa range; analysis of epit-ope by competition assays, sandwich assays.

Protein pathways: Direct analysis of complex stoichiome-try for the complexes: AMPk, CDC42-SopE, thymidine K, ATP synthase, ferritin complex.

Supershifting: The supershifting application consists in determining the presence of a subunit in a complex by MALDI high-mass mass spectrometry using monoclonal antibodies. An example of a supershifting experiment will be presented with the complexes formed by the fusion pro-teins GST-SopE and GST-CDC42 using a monoclonal antibody anti-GST.

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Screen for Arabidopsis thaliana Mutants Resistant to Agrobacterium tumefaciens—Mediated TransformationL. Pierre-Charles, K. Mir, T. Muth; Brooklyn College, Brooklyn, NY, United States.

Agrobacterium tumefaciens is a typical soil bacterium that causes crown gall disease in a variety of plant species. A. tumefaciens is capable of recognizing wound sites on a plant by detecting chemicals produced during the wound response of the plant. Laceration of the plant tissue causes the production of phenols and sugar molecules, which in turn trigger not only the chemotaxis of the bacteria towards the injury, but the processing of the tumor-inducing plasmid (Ti plasmid) as well as the expression of certain virulence genes. It is known that the bacterim inserts a segment of its Ti plasmid, the transfer DNA (T-DNA), into the plant at the wound site. The integration of the T-DNA allows for the production of certain plant hormones. It is the uncon-trolled plant cell division stimulated by the overproduction of growth hormones that leads to the formation of tumors. Through experimental analysis we seek to identify mutant lines of Arabidopsis that are resistant to transformation, or hypersensitive to transformation. Identification of resistant or hypersensitive plant types is done through root tumor assays as well as GUS assays. In using these methods, it was determined that mutants show evidence of increased resistance while other mutants exhibit susceptibility to transformation similar to wild types. There is little known about the mechanism behind A. tumefacien attachment and transfer of T- DNA into plant cells, and it is our hope that identification of Arabidopsis genes associated with transfor-mation in this screen will allow greater understanding of how the bacterial genetic material is transferred into the plant genome.

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Investigating Assembly of HIV—Gag Protein with Oligonucleotides using a Quartz Crystal Microbalance with Dissipation MonitoringJ. Simpson1, A. Jaiswal2, A. Stephen1, S. Datta3, A. Rein3, R. Fisher1; 1Protein Chemistry Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, United States, 2Q-Sense, Inc., Glen Burnie, MD, United States, 3HIV Drug Resistance Program, NCI-Frederick, Frederick, MD, United States.

The structural protein group-specific antigen (Gag) is the only viral product required for retrovirus assembly and will form virus-like particles (VLPs) in vitro in the presence of nucleic acids. The nature of the contribution of nucleic acid to particle assembly and structure is not fully understood. Further, interactions of Gag protein with nucleic acids have not been characterized in any detail. We have utilized a quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate the possible assembly of Gag particles in the presence of three oligonucleotides—TGx2.5, TGx10, and TGx20 (a 5-mer, 20-mer, and 40-mer, respectively). QCM-D provides information on both mass changes, measured as a change in frequency, and structural changes measured as changes in energy dissipation.

TGx2.5, TGx10, and TGx20 were immobilized, via a biotin linker, to an amine-coupled NeutraAvidin (Pierce Chemical Co., Rockford, IL) surface. HIV Gag protein was then passed over the surfaces via a peristaltic pump. QCM-D data are summarized as follows: The frequency values decreased with time when Gag was flowed over the oligo surfaces, indicating binding of protein. A continuous decrease in dissipation and frequency values was observed during the binding of Gag protein with the longest oligo-nucleotide, TGx20, indicating increase in the rigidity of the oligo-protein film. This suggests the formation of inter-molecular complex(es) between the bound oligo and the protein. Binding of Gag with TGx10 and TGx2.5 showed varying dissipation values with time. This indicates oligo-protein assemblies go through several structural changes during the course of binding. The control NeutraAvidin surface exhibited an increase in dissipation on binding with Gag that is typical of mass adsorption, indicating an increase in the viscoelasticity of the adsorbed film.

This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Funded by NCI Contract No. N01-CO-12400.

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The ABI 3730 DNA Analyzer is an Efficient System for Simultaneous, Bi-Directional Analysis of Dual-Labeled Probes Used to Study Protein-DNA InteractionsG. L. Spangler; Howard Hughes Medical Institution/Dept. of Molecular Biology and Genetics, Baltimore, MD, United States.

DNA footprinting is a powerful tool to study regions of DNA where proteins bind. Traditionally, target DNA fragments are labeled with 32P, incubated with the protein



of interest, then subjected to chemical nuclease attack. The DNA probe is then purified and nicked fragments analyzed on sequencing gels to reveal regions of DNA protected by the protein from attack.

The traditional method is a time-consuming and dif-ficult protocol requiring specialized skill. Proteins with low dissociation constants require an additional preparatory separation before analysis, which reduces the yield of bound probe. The isotope is labile, involves safety considerations, and requires a separate labeling and purification step. The finished gels must be dried down and exposed. This adds time to the overall process, varies according to the potency of the isotope, and decreases resolution. Normalization and alignment of free and bound probes is difficult. The one-dimensional nature of the single label necessitates separate analysis of each DNA strand, and the analytical gel limits the length of the analytical window.

We have developed a novel method of DNA footprint-ing that utilizes the multi-dimensional nature of fluoro-phores to label both strands of the DNA target, which per-mits differential analysis of both strands simultaneously. Labeled primers are commercially available for use as PCR primers, permitting simultaneous labeling and amplifica-tion of the DNA probe. The use of the chemical nuclease 1,10-phenathroline-copper facilitates in-gel attack of free and bound probe, which increases the yield of bound probe and reduces perturbation of the three-dimensional confor-mation of the complex. Samples can be analyzed rapidly by standard automated sequencing instrumentation with baseline, base pair resolution. Software data manipulation reduces normalization issues. The result is high-quality data acquisition in a matter of hours instead of days.

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Measuring the Stoichiometry and Cooperativity of HIV Gag Binding to Nucleic Acids by Surface Plasmon Resonance SpectroscopyA. G. Stephen1, S. Datta1, K. M. Worthy1, L. Bindu1, K. Turner2, D. Fabris2, A. Rein1, R. Fisher1; 1NCI-Frederick, Frederick, MD, United States, 2University of Maryland Baltimore County, Baltimore, MD, United States.

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA) and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)n oli-gonucleotides using surface plasmon resonance spectros-copy. A single NC protein is able to bind to more than one immobilized oligonucleotide. However, if oligonucleotides are immobilized at low densities (~10RUs), then NC no longer displays this behavior. As NC is proposed to be the nucleic acid binding domain of Gag, we would expect Gag to show the same behavior. To investigate the stoichiometry and cooperativity of Gag binding to oligonucleotides, we must ensure that Gag is unable to bind to two oligonucle-otides at once. In addition, we need to know accurately how much oligonucleotide is immobilized so we can cal-culate the stoichiometry of Gag binding. We developed a

method where we can determine precisely the amount of oligonucleotide immobilized at very low-density surfaces (0.1–2 RUs). In this approach, we used electrospray ion-ization Fourier transform mass spectrometry to determine that two and four molecules of NC bind to d(TG)5 and d(TG)10. We were then able to use NC injections to cali-brate the immobilized oligonucleotide and determine an accurate measurement of the surface density. Using this approach, we have measured the binding of Gag to d(TG)n. Gag binds to a 5-mer and a 20-mer with a stoichiometry greater than 1 and 4, respectively. This suggests that once Gag is bound to the immobilized oligonucleotide, addi-tional Gag molecules bind to the Gag already bound to the oligonucleotide, forming oligo-Gag-Gag complexes.

P94-S

Fishing for RNA-Binding Proteins from the HeLa Cell Nuclear Extract Using Biacore 3000S. P. Yadav, G. C. Shukla; The Lerner Research Institute, Cleveland, OH, United States.

Biacore’s surface plasmon resonance technology is widely used in the study of affinity and kinetics of interac-tions between two or more biomolecules, receptor-ligand interactions, and in understanding the kinetics of bio-materials. The analyte protein bound to the immobilized ligand on the sensor chip can be recovered for subsequent identification by mass spectrometry. Earlier studies have demonstrated that Biacore can be used to fish out proteins from crude mixtures in a highly purified state, and the recovered protein can be identified by upstream mass spec-trometry. However, earlier studies have been done mainly for the known target protein. The study of RNA-binding proteins is very important for understanding the assem-bly and function of RNA-protein complexes in the splice-osome. In the current study, a biotinylated short single hair-pin loop RNA was immobilized on the streptavidin sensor chip, and the HeLa cell nuclear extract was passed over it to identify the proteins bound to the biotinylated RNA stem-loop. The results of recovered analytes identified by mass spectrometry show that the Biacore 3000 could be a useful technique to identify previously unknown RNA-binding protein/s from nuclear extracts.

mass spectrometry

P95-M

Mass Spectrometric Proteomic Profiling of Biological Samples using Sample Preparation On Chip (SPOC)C. M. Belisle1, C. Menzel2, J. K. Mirshad1, J. A. Walker2, I. Chen2, K. Steinert2, U. Roth2; 1QIAGEN, Freemont, CA, United States, 2QIAGEN, Hilden, Germany.

Proteomic profiling of biological samples using mass spectrometry has been a goal for biomarker discovery and disease research for some time. It remains the current chal-


JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007 ��

lenge to reliably and reproducibly isolate and to sequence the potential biomarkers found in high-throughput screen-ing efforts by tandem mass spectrometry (MS/MS) to dif-ferentiate the important and meaningful biomarkers from ambiguous pattern artifacts. To this end, top-down and bottom-up proteomic workflows have yielded advantages and disadvantages, depending upon implementation into the research field.

We report on our recent optimization of protocols and MALDI-MS chip design to apply a sample preparation on chip (SPOC) technology for the profiling of serum sam-ples. This technique facilitates an efficient, simple, and reproducible isolation of proteins/peptides combined with the possibility of effectively concentrating low-abundance molecules. The benefit of further up-front sample fraction-ation is demonstrated with the removal of high-abundance proteins via immuno-depletion columns or ultra-filtration. The utility of the chips for direct identification of the iso-lated proteins from serum using latest generation TOF/TOF technology will also be presented.

P96-T

Characterization the Sequence and Post-Translational Modification of Large Peptides by Tandem ETD MS/MSA. Brekenfeld, C. Bäßmann, R. Hartmer; Bruker Daltonik GmbH, Bremen, Germany.

Protein/peptide analysis is commonly based on use of a tandem mass spectrometer combining electrospray ioniza-tion and collision-induced dissociation (CID). The major disadvantage of collisional ion activation is the internal heating of the parent ion, which predominantly yields cleavages of the weakest bonds, resulting in less informa-tive fragment spectra; this often limits peptide sequence determination.

To better elucidate the peptide structure, more selec-tive fragmentation techniques are of huge interest. Electron transfer dissociation (ETD) in a non-linear Paul trap has been introduced as a new fragmentation technique, which avoids internal parent ion heating. Induced by the electron transfer, the intermediate peptide radical cations fragment randomly at each amino acid position of the peptide back-bone, which is particularly suitable for the sequence char-acterization of larger peptides and for post-translational modification (PTM) identification.

We investigated the electron transfer dissociation of large peptides using the Bruker HCTultra PTM Discov-ery System. Multiply charged positive peptide target ions are generated via conventional electrospray or via off-line nanospray. Electron transfer dissociation of larger peptides is done in a non-linear three-dimensional Paul trap, where consecutive trapping of peptide cations as well as reagent anion accumulation is enabled under full automatic soft-ware control.

Common resonance precursor activation via CID MS/MS almost always results in poor sequence information if the peptide molecular weight exceeds 3000 Da. ETD MS/MS of the same precursor ion provides much better sequence information. Based on the enhanced resolution of the utilized instrument, since even quadruply charged frag-

ment ions are identified, the entire amino acid sequence is read out of the ETD MS/MS spectrum.

Even when larger PTM peptides were investigated under ETD conditions, labile bounded PTMs remain attached to the peptide backbone, and are localized without ambigu-ity. ETD MS/MS data of larger phosphorylated as well as glycosylated peptides will be presented.

P97-S

A High-Throughput Method for the Characterization of Nuclear Receptor DynamicsM. J. Chalmers, S. A. Busby, S. Prasad, J. B. Bruning, B. D. Pascal, M. R. Southern, P. R. Griffin; Scripps Florida, Jupiter, FL, United States.

Introduction: Nuclear receptor transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) are the molecular targets of drugs for the treat-ment of a wide range of diseases. Ligand-induced changes in structure/dynamics of the receptor drive dissociation of co-repressors, heterodimer formation, co-activator binding, and subsequent transcriptional activity. Characterization of the dynamics of the receptor is critical to understanding the mechanism of activation.

Methods: Hydrogen/deuterium (H/D) exchange is a technique for measuring protein dynamics. Here we describe a two-part approach to generate a high-throughput (<1 h/compound) H/D method to measure changes in the dynamics of the receptor ligand-binding domain (LBD). First, a comprehensive set of differential H/D data are acquired with a number of well-characterized ligands (~24 h/compound). From these data, a single period of H/D on-exchange is chosen that provides the maximal distance between dynamics of ligand-bound and apo-receptor. Next, the chromatography is optimized for the rapid elution of a subset of peptides that span the LBD. To aid this step, we incorporated 1.9-µm stationary phase HPLC columns into our H/D exchange platform. Single time point H/D exchange experiments are then used to probe the dynamics of the receptor.

Results: Comprehensive H/D exchange experiments were performed with five ligands that induce varying degrees of PPARγ activity (rosiglitazone, MRL-20, MRL-24, nTZDpa, BVT.13). For all regions of the protein, 60 sec on-exchange provided the maximum difference between the apo- and ligand-bound receptor. Fifteen peptides were selected that span the LBD. A chromatography method was developed that allowed for the separation of the peptides of interest in <10 min. We then proceeded to characterize 27 ligands in a 24-h time period.

Conclusions: We have developed an H/D exchange method for rapid characterization of ligand binding to PPARγ LBD. The approach is suited to the analysis of other nuclear receptors.


�� JournalofBiomolecularTechniques,Volume18,issue1, feBruary2007

P98-M

Class Imaging—Multivariate Statistical Classification of MALDI Tissue Imaging DataS. Deininger1, M. Schuerenberg1, C. Luebbert1, A. Fuetterer1, M. Gerhard1, D. Suckau1, C. Roecken2, M. Ebert3, A. Walch4; 1Bruker Daltonik GmbH, Bremen, Germany, 2Charite University Hospital, Berlin, Germany, 3Technical University Munich, Munich, Germany, 4GSF Institut fuer Pathologie, Neuherberg, Germany.

MALDI imaging is a powerful tool that allows the acquisition of tissue-specific mass spectra with spatial reso-lution. Here we applied statistical tools to generate infor-mation from MALDI imaging datasets. Unsupervised principal component analysis (PCA) is discussed to access tissue-specific variance in the data, and a supervised clas-sification approach is used to classify specific tissue types.

Methods: Tissue cryosections were thaw mounted on conductive coated glass slides, and the MALDI matrix was applied. Data were acquired on a MALDI-TOF mass spectrometer in linear mode. Statistical analyses were per-formed with the ClinProTools 2.1 software (Bruker Dal-tonics). Either the peptide masses detected by PCA were displayed in the MALDI images, or the principal compo-nents from the classification as such were used to visual-ize tissue classes rather than the distribution of molecular weights across the tissue.

Results: Unsupervised PCA analysis showed that the variance in the MALDI imaging datasets reflected the variance in the tissue. One limitation of the PCA was that on tumor sections from different patients, the vari-ance between the patients was high, and therefore the PCA reflected largely the difference between the patients rather than variance in individual tissues.

Using supervised classification approaches, it was pos-sible to classify characteristic tissue types and to apply these classifications to unknown tissue. This approach opens the door for diagnostic applications.

P99-T

A Simple Method to Quantitate Selenomethionine Incorporation into ProteinsL. A. Fabrizio, P. Tempst, H. Erdjument-Bromage; Memorial Sloan-Kettering Cancer Center, New York, NY, United States.

Selenomethionine substitution is the preferred method for preparing heavy-atom derivatives of proteins for crystal structure determination using the multi-wavelength anom-alous diffraction phasing method. This approach allows researchers to take advantage of the anomalous signal from a number of diverse atoms. We recently published a pro-tocol describing a number of variables that play a role in determining incorporation efficiency of selenomethionine into mammalian expression systems.1 Here we describe, in detail, a simple method for assessing selenomethionine sub-stitution by replacement of methionine in homogeneous protein preparations. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) technology following trypsin proteolysis of the recombinant protein, we are able to evaluate variables that play roles in affecting

selenomethionine incorporation. Examples will illustrate (a) the ease of identification of modified peptides contain-ing the selenomethione and (b) relative quantitation of such peptides when compared with the control, unmodi-fied peptides.

reference

1. Barton WA, Tzvetkova-Robev D, Erdjument-Bromage H, Tempst P, Nikolov DB. Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells. Protein Science 2006;15:2008–2013.

P100-S

Analyses of In Vitro Non-Enzymatic Glycation of Normal and Variant Hemoglobins by MALDI-TOF Mass SpectrometryS. Gupta, L. Vida, G. Honig, S. Krishnanchettiar, S. Lateef, G. Jayathilaka, B. Lee; UIC, Chicago, IL, United States.

MALDI-TOF MS is used successfully in investigating in vitro glycation of normal and variant hemoglobins (Hbs). Singly glycated, doubly glycated, and/or multiply glycated glycoisoforms of the alpha-globin, beta-globin, and gamma-globin of Hbs are observed. Different glycation rates are observed for normal and variant Hbs, with the normal Hb A having the slowest rate. The normal Hb A is more stable than the Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego upon condensation with glucose at 37°C. Data reveal that with longer incubation time (up to 5 d), higher glucose concentration (up to 1 M), and higher temperature (up to 37°C), the number of glycated amino acid residues of Hbs increase. The extent of the glycation of both Hb A and Hb F increases upon changing the solvent from PBS (pH 7.4) to carbonate buffer (pH 10). However, this pH change has a lesser effect on the glycation of the Hb C, Hb E, or Hb Leiden. In this study, higher concentration of the glucose is used to increase the reaction rate of the less reactive residues, and higher pH is used to alter the structures of proteins and consequently expose the reac-tive amino acid residues. However, incubation time is kept relatively short. Results obtained in this study could lead to the inference that effects of the incubation time, glu-cose concentration, temperature, and pH on the linkage of Hbs with glucose would be similar under the diabetes conditions in vivo (~37°C, ~neutral pH, ~0.007 M glucose concentration). This conclusion agrees with our previous report on glycation of all the peptides/proteins that we have studied with glucose.

P101-M

New Setup for Top-Down Characterization of Proteins via Consecutive Ion/Ion ReactionsR. Hartmer, M. Lubeck, C. Bäßmann, A. Brekenfeld; Bruker Daltonik GmbH, Bremen, Germany.

The conventional MS “bottom up” strategy for protein identification is the tandem MS/MS analysis of tryptic digests of complex proteins mixtures. Via enzymatic diges-



tion, even larger proteins are cut into smaller peptides. In order to reduce the resulting complexity of the tryptic digest, additional chromatographic separation is used in front of the tandem MS/MS analysis. The bottom-up strat-egy, however, has shown its limitation when the character-ization of multiply post-translational modified protein is of interest. The unique identification of several individual peptides from a tryptic digest does not provide the needed information if the interaction of the different modifications of the intact protein is of primary interest.

Electron transfer dissociation (ETD) is particularly dedicated for the sequence analysis of larger peptides as well as for the identification of PTMs, as weakly bonded PTMs survive the prompt odd-electron-driven fragmentation of the electron transfer. However, if a multiply charged protein (z > 10) is analyzed via tandem ETD MS/MS, the resulting fragmentation spectrum shows a highly complex mixture of multiply charged fragment ions (zfragment ion = 1–9).

We have modified the nCI source as well as the acqui-sition software of the HCTultra PTM Discovery System to allow the selective and consecutive ion accumulation of different types of reagent anion. For the initial ETD step, odd-electron reagent anions are selectively accumulated, and for the subsequent proton-transfer reaction, even-elec-tron reagent anions revealing a strong proton affinity are accumulated. The second PTR step is necessary to signifi-cantly reduce the complexity of the above-mentioned ETD MS/MS-data, particularly when highly multiply charged proteins are investigated. The final charge stripping step produce a mixture of 1+, 2+, 3+, and 4+ fragment ions. With the present setup we were able to characterize the amino acid sequence of the intact proteins (e.g., ubiquitine, cytochrome C).

P102-T

Electron Transfer Dissociation, Multi-Stage Activation, and Neutral Loss Initiated MS3 Utilized in the Characterization of Phosphorylation Sites of Human KinasesM. P. Hornshaw1, N. Morrice2; 1Thermo Electron, Hemel Hempstead, United Kingdom, 2MRC Protein Phosphorylation Unit, Dundee, United Kingdom.

The standard mass spectrometric technique for the analysis of phosphopeptides on a linear ion trap mass spectrometer is to perform MS/MS followed by MS3 of the putative neutral loss peak observed by MS/MS. This is usually termed data-dependent neutral loss MS3 (DDN-LMS3). The strength of this approach is the sensitivity in full scan MSn of linear ion trap technology and the char-acteristically strong neutral loss peak, which flags the MS/MS spectrum as potentially belonging to a phosphopep-tide. This approach has been much applied to phospho-

proteomic analyses typically involving up-front phospho-peptide enrichment, such as TiO2 chromatography. Indeed, a recent article from Mann and co-workers identified 6600 phosphorylation sites utilizing a hybrid linear ion trap Fou-rier transform mass spectrometer. However, there are also weaknesses, such as the fact that phosphotyrosine residues rarely produce a neutral loss from the precursor during MS/MS.

Recently, a new dissociation technique, electron trans-fer dissociation (ETD), has been invented in the lab of Don Hunt. ETD has different but complementary characteris-tics compared to CID. For example, (1) ETD works best on multiply charged ions of 3+ and above, whereas CID works best on 1+ to 3+ ions; (2) amino acid side chains and important modifications such as phosphorylated amino acid residues are left intact by ETD, producing a rich lad-der of c and z ions for sequencing. CID produces mostly b- and y-type ions by collision with gases.

Work reported here compares DDNLMS3 data (and another neutral loss–based technique referred to as multi-stage activation, also utilized in the work of Mann and colleagues) generated on a series of human kinases with results obtained by data-dependent ETD. The different dissociation techniques were found to be complementary, and a number of novel phosphorylation sites were identified on such well-known kinases as PKB and SAPK2a.

P103-S

Rapid Nanobore UPLC Separations Coupled with ESI MS/MS for Improved High-Throughput Protein IdentificationT. McKenna, I. Campuzano, I. Ross, A. Wallace, J. I. Langridge; Waters Corporation (MS Technologies Centre), Manchester, United Kingdom.

Mass spectrometry has established itself as the primary technique for identifying proteins due to its unparalleled speed, sensitivity, and specificity. Strategies for digestion of the proteins use a specific protease that cleaves at predict-able residues along the peptide backbone, providing smaller stretches of peptide sequence more amenable to mass spec-trometry analysis. When coupled with protein-level pre-fractionation strategies, such as one-dimensional PAGE, thus reducing the complexity of the protein mixture, this approach has proven highly successful in comprehensive protein identification and characterization. The downside of this approach is the number of gel samples, or fractions, to be analyzed by the LC-MS/MS system. With typical analytical HPLC run times of 45 min to 1 h, the amount of time required to analyze one top-level sample can be prohibitive.

Here we describe the use of elevated flow rates combined with nanoscale columns packed with sub-2-µm particles for rapid separations using a nanoUPLC system. Increasing the flow rate to 900 nL/min and running a very rapid gradi-ent over 8 min on a 75 µm × 15 cm column, allows high-quality peptide separations to be achieved with a sample-to-sample inject time of 10 min. This, combined with an orthogonal acceleration time-of-flight mass spectrometer, using a newly developed high-speed data-dependent MS/MS approach fragmenting up to eight precursor ions per second, allows for the rapid characterization of simple pro-tein mixtures, such as those obtained from 1D gel bands.

We will present data from standard tryptic digests of known proteins and simple mixtures of protein digests used in the development of this method, and data from in-gel digests of 1D gel bands.



P104-M

Comparison between Sample Preparation On Chip (SPOC) and Off-Line MALDI-MS Sample-Preparation Approaches for Sample Clean-Up and IMAC EnrichmentC. Menzel1, U. Roth1, J. Mirshad2, J. A. Walker2, I. Chen2, K. Steinert1, C. Belisle2; 1QIAGEN, Hilden, Germany, 2QIAGEN, Freemont, CA, United States.

Optimized sample preparation is crucial for successful and sensitive detection of peptides by MALDI-TOF MS. In particular, the protein identification of low femtomole amounts of analyte requires further clean-up and concen-trating steps to acquire significant spectra. This aspect is even more pronounced if the analysis of post-translational modifications is the goal. A prominent example is the map-ping of protein phosphorylation sites, which is often ham-pered by the lack of phosphopeptides in spectra of crude digests. For that reason, very often micro-columns filled with reversed-phase material for clean-up, or IMAC or TiO2 resins for phosphopeptide enrichment, are employed prior to MALDI-MS analysis.

The performance of these off-line technologies will be compared to a new sample preparation on chip (SPOC) technology. The SPOC technology integrates all steps of sample preparation directly on the MALDI same-sample support by means of a patterned surface forming rings of different wetability and chemical functionality.

The superior performance of this SPOC approach for sample desalting and concentrating of in-gel digests will be presented. Moreover, the analysis of protein phosphoryla-tion using chips with IMAC functionality is compared to off-line column or bead–based strategies with respect to the efficiency and specificity of phosphopeptide enrichment.

P105-T

Peptide Sequence Validation using Retention Time InformationP. Metalnikov, A. Pasculescu; Samuel Lunenfeld Research Institute, Toronto, ON, Canada.

The increasing number of MS/MS spectra, generated by new mass spectrometers with fast scanning (LTQ, Ther-moElectron, etc.), requires better and more robust spectra filtering. Retention time is one of the important peptide characteristics, but it is not used very often with the aim of peptide sequence validation. Here, we present an approach to filter Mascot (Matrixscience) search results on the basis of comparison of experimental retention times with pre-dicted ones.

Proteins were digested in-gel or in-solution with tryp-sin according to conventional protocols. Resulting peptide mixtures were analyzed on line with an LC-MS system (HP 1100 Nanoflow HPLC [Agilent] and LTQ Mass Spec-trometer [ThermoElectron]). Peptides were separated on custom-packed 75-µm i.d., 10-cm-long PicoTip columns (New Objective) packed with 3.5-µm C18 beads (Pursuit, Varian). The column effluent was sprayed directly into the transfer tube of the mass spectrometer. The Sequence Specific Retention Calculator (http://hs2.proteome.ca/SSRCalc/SSRCalc.html) was used for calculation of pep-

tide relative hydrophobicities. This program uses a refined model for determination of hydrophobicity, presented recently by Oleg Krokhin (University of Manitoba). Two columns are automatically added to the standard Mascot peptide summary page: scan number (reflecting retention time) and relative hydrophobicity. The plot of scan num-ber vs. hydrophobicity is also created. The linear regres-sion could be calculated with a subset of “good peptides” (having high Mascot score, e.g., >50). Based on this regres-sion, predicted scan numbers could be identified. The rest of the peptides (with score <50) are tested, and sequences with scan numbers too far from predicted are rejected as false-positive hits. The algorithm was trained on standard digests such as BSA, and is now applicable to any LC-MS Mascot search results. Rejected hits were confirmed by manual inspection of MS/MS files. It appeared that this script is especially useful for validation of singly charged peptides, which often give fragmentation spectra of poor quality.

P106-S

Microwave-Assisted Enzymatic CleavagesV. Pham, W. Sandoval, D. Arnott, J. Lill; Genentech, South San Francisco, CA, United States.

Previously we reported on a protocol for the complete de novo sequencing of a therapeutic monoclonal antibody. We are continuously evaluating biochemical, analytical, and bioinformatics methods to add to our toolbox for increased throughput. Microwave-assisted proteomics has recently emerged as a way to decrease biochemical and pro-teolytic reaction time. Here, we describe an investigation into microwave-assisted proteolytic cleavage using trypsin, Lys-C, and Asp-N. Microwave irradiation was also applied to the digestion of proteins for removal of N-linked sug-ars using PNGase-F and removal of pyroglutamyl group employing pyroglutateamino peptidase (PGAP).

Method: Proteins were digested with trypsin, Lys-C, Asp-N in the thermocycler at 37°C for 5, 10, and 30 min, or in the CEM microwave at 37°C, 2 W, at the same time points. Generated peptides were then analyzed by SDS-PAGE for quantitation or via LC/MS/MS for sequence cov-erage. Microwave-assisted deglycosylation of proteins was performed at 37–60°C for 2 min to 1 h or in the thermo-cylcer at 37°C for 2 min to overnight, and molecular weight analysis was performed. PGAP digestion was performed in the thermocycler or microwave at 90°C at different time points, and proteins were sequenced by Edman degradation analysis to assess de-blocking of the N-terminus.

Preliminary Results: For proteolytic digestion, a sig-nificant reduction in incubation time was observed for trypsin when microwave irradiation was applied. However, no benefit was observed with microwave-assisted Lys-C or Asp-N digestion. Deglycosylation of proteins using microwave irradiation markedly decreased the incubation time from several hours to 10 min for most proteins. The initial deblocking using PGAP occurred more rapidly in the microwave versus thermocycler, but overall incubation times were not significantly reduced.



P107-M

A Workflow to Enable Intelligent and Targeted Second-Chance in High-Throughput Mass Spectrometry ExperimentsA. Prakash1, D. Robinson2, J. Sutton1, A. Zumwalt1, M. Lees3, L. Bonilla1, N. Kelleher2, M. Senko1; 1Thermo Fisher Scientific, Cambridge, MA, United States, 2University of Illinois, Urbana, IL, United States, 3Advion Biosciences, Ithaca, NY, United States.

In a typical mass spectrometry experiment, the state-of-the-art sequencing algorithms are able to identify only about 10% to 20% of the tandem-MS spectra. Moreover, due to the high dynamic range and complexity of a sample like human plasma, many proteins or peptides do not even lead to a MSn spectra. This list includes the differentially expressed markers in a typical biomarker discovery study, features that follow a particularly interesting trend, time-course studies, etc. While many of such features can be flagged using computational analysis of the MS1 signal, MSn spectra are still required to get an annotation.

Using an in-line post-column split with the TriVersa NanoMate (Advion Biosciences, Ithaca, NY) coupled with the highly sensitive Orbitrap, we have built a system where we collect LC fractions in a 96-well plate, which are then available for direct infusion at a later time. This gives the advantage of direct infusion on a sample of much lower complexity, thus providing longer analysis time, which in turn enables us to (1) perform intelligent and targeted iso-lation and fragmentation, (2) attempt different fragmenta-tion methodologies, such as CAD, ETD, IRMPD, etc., and (3) perform multi-stage MSn. This analysis is fully auto-mated, and thus capable of targeting hundreds of features. As a result, given a feature list (similar to an inclusion list), which can be built using differential analysis software like Sieve, our workflow provides a targeted and iterative re-analysis in a high-throughput fashion to get high-quality tandem spectra, and thus a much higher chance of getting an annotation.

P108-T

Distribution of Tarceva in Pancreatic Tumors by Imaging Mass Spectrometry Using a Finnigan LTQ Linear Ion Trap Mass Spectrometer with vMALDI SourceM. C. Prieto Conaway1, S. Beachy2, R. Pitoniak2, B. Hylander2, P. Wang2, K. Marlar2, E. Repasky2, L. Kazim2; 1ThermoFisher Scientific, San Jose, CA, United States, 2Roswell Park Cancer Institute, Buffalo, NY, United States.

Imaging Mass Spectrometry (IMS) using matrix-assisted laser desorption ionization (MALDI) is being investigated using a linear ion trap with the overall goal of analyzing the distribution of various targeted therapies within patients’ tumors. The drug chosen for evaluation here is erlotinib (Tarceva) which targets the epidermal growth factor receptor (EGFR), a type I receptor tyrosine kinase (TK) involved in cellular differentiation and prolif-eration, by binding to the ATP pocket and inhibiting the autophosphorylation of the receptor. Tarceva has demon-

strated clinical activity in non-small cell lung cancer, head and neck cancer, and ovarian cancer in Phase II studies. The sensitivity and MSn capabilities of the Finnigan LTQ are exploited for the unambiguous determination of the distribution of this drug within human pancreatic tumors. Custom software was used to acquire data while rastering the tissue and data visualization software used to display the 2- and 3-dimensional images.

SCID mice bearing patients’ pancreas tumors grown as xenografts were dosed once with 2.5 mg Tarceva in 6% Captisol, or Captisol alone, delivered by oral gavage. Six-teen hours following the administration of treatment or the vehicle, the pancreas tumors were resected, snap fro-zen immediately, and then 10-µ-thick tissue sections were prepared with a cryotome and placed on various MALDI surfaces. Nebulized matrix was applied with a commercial airbrush. Peaks corresponding to the m/z of both Tarceva and its O-demethylated metabolite, OSI-420, were found distributed in Tarceva-treated tumor, but not in the con-trol sample. Results from single reaction monitoring (SRM) experiments on the drug precursor at m/z 394 and the metabolite at m/z 380 were mapped to visualize the drug distribution within the tissue. The results confirm that the Finnigan LTQ with vMALDI source mass spectrometer can be used to localize Tarceva and its metabolite in a xeno-graft tumor model.

P109-S

Improved Identification of Membrane Proteins by MALDI-MS/MS Using Matrix Pre-Deposited MALDI ChipsU. Roth1, C. Menzel1, A. Poetsch2, D. Schlüsener3, M. Rögner2, K. Steinert1; 1QIAGEN, Hilden, Germany, 2University of Bochum, Bochum, Germany, 3University of Bochum, Bochum, Bochum, Germany.

Due to their low abundance and limited number of trypsin cleavage sites, integral membrane proteins are noto-riously difficult to identify and to analyze by mass spec-trometry. Our approach to this problem is based on a novel technology for peptide sample preparation that uses plates with pre-deposited matrix spots of sub-micron size CHCA crystals prepared by vacuum sublimation onto an ultrap-hobic surface (Mass·Spec·Turbo Chips, QIAGEN). Such a design enables highest sensitivity analysis and reproducible and automatable sample preparation, suitable for both gel- and LC-based MALDI-MS approaches.

In order to compare the chips’ performance with that of standard stainless steel target plates, different sample amounts of two model membrane proteins—bacteriorho-dopsin and subunit A of ATP synthase—were separated by SDS-PAGE and in-gel digested with trypsin. After diges-tion, aliquots were spotted either as classical dried droplet preparations on stainless steel plates using CHCA as matrix, or applied directly onto the matrix spots pre-deposited on the chip surfaces. Spectra were acquired on Voyager DE-Pro or 4800 MALDI-TOF instruments (Applied Biosys-tems) and protein identification was performed by peptide mass fingerprint or MS/MS ion search using the Mascot algorithm (Matrix Science). The results were evaluated by comparing S/N ratios, sequence coverage, Mascot scores



and the total ratio of protein identifications vs. unsuccess-ful attempts. Analyses of the spectra obtained revealed a significantly larger number of peptide signals using the chip approach, which resulted in increased sequence cover-age and more reliable protein identifications.

P110-M

A New Preparation Technique for MALDI Tissue ImagingM. Schuerenberg, C. Luebbert, S. Deininger, R. Mueller, D. Suckau; Bruker Daltonik GmbH, Bremen, Germany.

The quality of MALDI Imaging analyses crucially depends on the matrix application protocols. Unfortunately, the currently used protocols have significant disadvantages that impair the robust and routine use of tissue imaging as analytical or even diagnostic approach. While pneumatic spray preparations provide high surface homogeneity and spatial resolution of the images, the process is manual and highly irreproducible. Depending of the degree of tissue wetting either the analyte molecules are badly incorporated into the matrix (too dry) or the spatial resolution is lost (too wet). Nano-spotting on the other hand provides qual-ity spectra but as a sequential process it is slow, spatial reso-lution is limited by the spot raster (typical >100 µm) and perfect alignment with the mass spectrometer is critical.

We introduce an entirely new approach that combines the advantages of above methods and eliminates the dis-advantages. In the new preparation device, matrix aerosol (< 50 µm droplets) is created by vibrational vaporization under controlled conditions that is gently deposited onto tissue sections. The entire microscope slide with multiple sections can be matrix coated in one setup. An optical sen-sor allows to monitor and to control all relevant prepara-tion parameters at real-time: deposition periods, intervals, matrix layer thickness, wetness, drying rate, etc. This QC process built into the preparation process provides highly reproducible preparations over several slides and at different days. No user intervention is required in the fully auto-mated process.

On tissue it was possible to achieve lateral resolutions down to 50 µm.

P111-T

A New MS Technique for Relative Quantification of Proteins with Isobaric LabelsW. Jabs, M. Lubeck, U. Schweiger-Hufnagel, M. Behrens, A. Brekenfeld, C. Baessmann; Bruker Daltonik GmbH, Bremen, Germany.

Due to their superior MSMS sensitivity and acquisition speed, ion traps are ideal mass spectrometers for proteomics. However collisionally induced fragmentation techniques suffer from the 1/3 mass cut-off currently used in these instruments. As a consequence amino acid information from low mass ions (immonium ions, b1- and y1- ions) is typically missed. In addition, quantitation using isobaric labeling techniques (e.g., iTRAQ) requires the detection of low mass reporter ions, usually from precursor ions above

400 Th. A newly developed fragmentation technique for Bruker HCT ultra ion traps provides extraordinarily broad fragment spectra down to low mass fragments. The useful-ness of this technique is evaluated for protein quantifica-tion using isobaric iTRAQ labeling.

Two mixtures of 15 standard proteins were prepared: Sample A contained equimolar amounts of all 15 proteins and Sample B the same proteins in different concentrations. The concentration difference was in the range of 2 orders of magnitude. Mixed together the samples represented dif-ferent protein regulations from 10-fold down to 10-fold up-regulated. Digest and labeling were performed according to the vendor’s protocol. In order to have an internal duplicate, sample B has been labeled with 114 and 116 and sample A with 115 and 117. The peptides were separated by nano LC. Injected peptide amount varied between 110 and 1100 fmol. The data acquired on an ion trap MS instrument were analyzed in respect of their reporter ion amount.

The new fragmentation technique for ion trap MS enables the detection of fragment ions of low masses from high m/z values and is therefore highly suitable for applica-tions like iTRAQ based protein quantification. The soft-ware used for this purpose turned out to be most capable.

P112-S

Increased Selectivity for the Ion Mapping of Synthetic and Endogenous Small Molecules from Tissue Sections using MS/MS on a MALDI Q-Tof Mass SpectrometerM. Snel, E. Claude, D. Kenny, R. Tyldesley Worster, T. McKenna, J. I. Langridge; Waters Corporation (MS Technologies Centre), Manchester, United Kingdom.

Imaging the spatial distribution of molecules in tissue using MALDI mass spectrometers is a rapidly developing technique. The acquisition of accurate mass data in this type of experiment can be hampered in axial MALDI Tof systems. Even small changes in sample position and laser energy in the source region of this type of mass spectrom-eter affect mass measurement accuracy and mass spectral resolution. Here, we show how the use of an orthogonal Tof MALDI mass spectrometer circumvents these problems by decoupling the MALDI source from the mass analyser.

Imaging data were acquired on a MALDI Q-Tof mass spectrometer. The tissue sections were mounted on a target plate and moved in a raster pattern relative to the laser. To reduce interference from the biological matrix and enhance specificity the instrument was operated in MS/MS mode, a quadrupole was used for precise precursor ion selection. The sensitivity of specific ions was further enhanced by synchronising the high voltage push of the Tof mass analy-ser with the arrival of ions of appropriate m/z in the accel-eration region.

MALDI imaging information has been obtained from thin sections of rat tissue from animals doped with drugs, e.g., the well studied D2/D3 dopamine receptor antago-nist Raclopride as well as from untreated animals. Data obtained on the spatial distribution of drugs/drug metabo-lites and endogenous species will be presented. Challenges and future directions with MALDI imaging sample prepa-ration are discussed.



P113-M

Voltage/Current Divider Effects on Nanoelectrospray Threshold Voltage and Electrospray StabilityG. A. Valaskovic, A. W. Perala, C. J. Toher; New Objective Inc., Woburn, MA, United States.

Most commercially available nanoelectrospray ioniza-tion (NSI) sources possess well-defined electrical ground points in the mobile phase flow path. Inclusion of a ground point eliminates possible safety hazards involving source high voltage. Most home-built sources, however, lack a specifically defined ground point; the ground point often resides in injection valves, auto-samplers, or chromato-graphic pumps. Poorly placed ground-points affect ana-lytical performance because high-voltage (HV) source cur-rent flows to this point. HV for on-line NSI is commonly provided through an electrode contacting the mobile phase upstream from the emitter. Current flow between HV sup-ply, ground point, and nanoelectrospray plume (e.g., the current that flows from emitter to inlet) engenders the functional equivalent of a voltage-divider circuit. Relative impedance between the ground point and HV supply, and the impedance between the HV supply and NSI plume, determines effective spray voltage at the emitter tip. If rela-tive impedance to the ground point is too low, effective voltage at the emitter sinks below the HV supply, affecting plume stability or even preventing spray formation; this obstacle is amplified in gradient elution because mobile phase surface tension changes during the analysis. Periodic or chaotic spray drop-out is a typical result. Plume forma-tion and stability with gradient elution will be investigated using zero-dead-volume, optically clear, platinum electrode assemblies for HV and ground connections. Figure 1 shows a 150 µm platinum wire for HV contact at the junction between 75 and 50 µm ID fused-silica tubing. HV con-tact is made through a 150 µm platinum wire. Minimum requirements of tubing dimensions (length, inside diam-eter) between HV and ground points, while minimizing NSI threshold voltage disturbance, will be determined.

P114-T

HPLC/MS of Pesticides and Metabolites in Brain Tissue using Microwave Sample Preparation and MS Specific ExperimentsB. Winnik1, B. T. Buckley1, E. Richfield2, M. Thiruchelvam1; 1Rutgers, The State University of New Jersey, Piscataway, NJ, United States, 2Robert Wood Johnson Medical School, Piscataway, NJ, United States.

Parkinson’s disease (PD) is a neurodegenerative disease that is difficult to diagnose because there are no specific diagnostic biomarkers. It cannot be diagnosed solely on pathology. A reliable animal model may assist in identifica-tion of novel biomarkers. Paraquat (PQ) has been identi-fied both epidemiologically and experimentally as a pos-sible risk factor for PD in humans and mice. It has been demonstrated to preferentially kill dopaminergic neurons in the substantia nigra in mice and rats and its effects have also been found to be persistent with an apparent half-life of ~ 28 days. Its neurotoxic affects have been increased with concomitant doses of MPTP or other pesticides. One of the keys to determine it’s efficacy as a model for PD is to measure its concentration and disposition effectively in the brain tissue of the animal.

In order to measure the concentration of PQ, a mass spectrometric measurement was developed that was sensi-tive enough to measure low concentrations in small regions of the brain. Tissue samples are typically 10-15 mg in size and the concentrations within the sample are approximately 0.1 to 1.0 ng/mg. In addition to the measurement of PQ, MPTP and its metabolite MPP+ also had to be measured for this work. The MS method had to be able both to iden-tify and quantify each of these analytes, unambiguously. The key was good sample preparation. Being able to sepa-rate the analyte from the brain tissue was not straightfor-ward. A microwave accelerated solvent extraction (MASE) method was used to prepare these samples for mass spec-trometric analysis. This was coupled with a centrifuge fil-tration cleanup of extract. We have also used microwave assistance for extraction of chlorinated pesticides (PCBs) and other similar compounds from matrices such as serum and plasma, but the brain tissue extraction was one of the more difficult methods.

P115-S

Mapping PTMs with Electron Transfer DissociationT. Zhang, Z. Hao, R. Viner, Y. Huang, A. Huhmer, K. Miller; ThermoFisher, San Jose, CA, United States.

Biomarker discovery relies heavily on DNA data bases to identify biomarker candidates. However, such data bases reveal nothing about the post-translation modifications (PTMs) that decorate a protein or peptide and contribute to their essential activity. Characterizing these modifications is a requirement for subsequent validation studies.

The most prevalent PTM is phosphorylation which impacts more than one third of all proteins in a cell. For biomarker studies that focus on secreted proteins, glyco-sylation is a universal characteristic of such proteins. For biomarker studies looking at the peptidome many of these



peptides have a disulfide bridge that constrains the peptide in a particular shape.

Although mass spectrometry (MS) with collisional-induced-dissociation (CID) of peptides has proven to be a powerful and sensitive technique for the identification of candidate biomarkers, it has limitations in mapping PTM sites because the PTM attachment to the specific amino acid residue is often lost. This is not the case with a new dissociation technique referred to as Electron Transfer Dis-sociation (ETD). ETD cleaves the peptide backbone, but unlike (CID) it leaves phosphoryl groups attached to their amino acid sites.

This works presents the application of ETD to the char-acterization of PTM sites. The application of alternating MS scans with CID followed by ETD on an LC time scale will be shown for the elucidation of PTM sites.

P115A-T

High Speed Nanoscale UPLC Separations Combined with Off-Line MALDI MS/MS for Peptide AnalysisJ. Langridge, E. Claude, M. Snel, K. Compson, T. McKenna; Waters Corporation (MS Technologies Centre), Manchester, United Kingdom.

MALDI MS/MS is now a standard technique for pro-tein identification and characterisation and this method has been shown to work well with mixtures of low com-plexity. In the analysis of biological samples considerable sample processing and chromatographic separation is cru-cial to limit the complexity and dynamic range presented to the MS. A common approach to protein prefraction-ation is 2D or 1D PAGE. The downside of this approach is that the complexity of the sample is still such that an LC-MALDI experiment is required, prior to analysis. With typical HPLC run times of 45 min to 1 h, the time required to analyze one top level sample can be prohibitive.

In this poster we describe the use of elevated flow rates combined with nanoscale columns packed with sub-2-µm particles for rapid peptide based separations using a nanoU-PLC system. Increasing the flow rate of the separation from approximately 300 nL/min to 900 nL/min and running a very rapid gradient over 8 minutes on a 75 µm × 15 cm col-umn allows high quality peptide separations to be achieved with a sample to sample inject time of under ten minutes. The eluent is combined with matrix solution and spotted directly onto MALDI target plates. This combined with an orthogonal acceleration time-of-flight mass spectrometer equipped with a 200-Hz MALDI source, allows for the rapid characterisation of simplified protein mixtures, such as those obtained from 1D gel bands.

We will present data from standard tryptic digests of known proteins and mixtures of protein digests used in the development of this method and data from in-gel digests of 2D and 1D gel samples. This will be compared and con-trasted against data acquired using conventional separa-tions and mass spectrometric analysis.

metaBolomics

P116-M

Metabolomic Database Formats; What Do We Save and How Do We Save It?B. T. Buckley, I. Yang, A. Brooks; EOHSI-BRTC, Piscataway, NJ, United States.

Metabolomic data come in many forms depending on what was collected. In a study looking for biomarkers of exposure to H2S, whole blood was collected from partici-pants and subsequently analyzed by GC/ITMS. The data from chromatograms were sorted using principal compo-nent analysis. A key portion of the chromatograph was used to demonstrate differences between no exposure, a low and high exposure. True difference mass spectra were used to differentiate urine samples from mice before and after they ocnsumed green tea. MS data from control subjects looking at MTBE exposure sensitivity, showed a time course differ-ence from a single subject, following exposure. All of this data came from mass spectral data points but the pattern may not be appropriate for storage in a database that should be searchable. The patterns created are the most effective



way to verify biological differences in conditions and iden-tify potential biomarkers, but is not useful for comparing results from one study to another. Data in the raw phase (mass spectral data) cannot be compared to other raw data (NMR) and cannot be linked directly to metabolic path-ways.

A comprehensive database would include data that was; searchable, linked to metabolic pathways and comparable across analytical methods. The ideal database would also contain all requisite study subject data such as; diet, age, gender and genotype. Until we can create the ideal data-base one relating raw data to either pathway or compound will have to suffice. This presentation will spotlight the dif-ferent types of data from the studies described above as well as direct infusion MS data. The raw data from mass spec-tra can be related to compounds in metabolic pathways by generating theoretical mass spectrum. The database storage decisions on what to store and how to store it will be the focus of this presentation.

P117-T

Metabolite Identification Utilizing an Ion Trap–Time-of-Flight (IT-TOF) Mass SpectrometerJ. M. Ginter, N. Masayuki; Shimadzu Scientific Instruments, Columbia, MD, United States.

Metabolomics has proven to be the next tool for under-standing biochemical processes. While groups have taken various approaches to metabolomics (targeted or global), identification still remains the overarching goal. The use of high end mass spectrometers is prevalent in the proteomics arena for identifying proteins and one can argue that high mass accuracy instruments would be just as beneficial for identifying low molecular weight metabolites as well.

Utilizing an ion trap–time-of-flight system, data will be presented that highlights the advantages of multiple stages of high accuracy MS when identifying unknown metabolites. The IT-TOF is able to produce MSn data as well as take advantage of the resolution and mass accuracy of a TOF instrument. A series of standard pharmaceuti-cals and their metabolites will be analyzed via LC-MS/MS, and the data subsequently analyzed using novel metabolite identification software utilizing multivariate analysis tools. Additionally, the use of metabolite prediction software such as Lhasa’s Meteor will be highlighted.

P118-S

Direct Infusion Mass Spectrometry for Biomarker Detection of an Unstable Pesticide in Cells and Tea Metabolites in PlasmaI. Yang1, L. M. Domico2, S. Sang3, C. S. Yang3, B. T. Buckley1; 1Bionomics Research and Technology Center (BRTC) at EOHSI, Rutgers University, Piscataway, NJ, United States, 2Dept. of Neonatology, Children’s Hospital of Philadelphia, Piladelphia, PA, United States, 3Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ, United States.

Direct infusion mass spectrometry (DIMS) is an essen-tial technique for measuring chemicals that form from deg-radation processes in real time. As an analytical tools for metabolomics and biomarker discovery, it can be used to directly link appearing and disappearing molecules to one another using proper MS monitoring experiments.

It was used in two cases where the analyte products were originally unknown. In the first, mancozeb (MZ), a fungicide was monitored for appearance inside neuronal cultures. The precursor ion could not be measured effec-tively so ion fragmentation of the parent compound was evaluated using full scan MS, tandem mass (MS2) and triple stage mass spectrometry (MS3). MZ was detect-able at levels near 10 nM as measured in the cell lysate. MS, MS2 and MS3 revealed fragmentation ions that were directly related to MZ and could be sued for quantative analysis. Quantitation of cell media and lysate revealed that approximately 7–8% of the original dose crossed the cell membrane and remained stable for approximately 3 h. The parent compound began to decompose and/or metabolize inside the cell but the product was detectable up to 14 h post-treatment.

In the second DIMS experiment, (-)-Epigallocatechin-3-gallate (EGCG) the most abundant and biologically active compound in tea, was monitored for it’s breakdown/oxida-tion product(s). The stability of EGCG in Tris-HCl buffer was investigated using real-time mass scanning combined with tandem mass ion mapping. An EGCG quinone prod-uct, EGCG dimer quinone and other compounds related to autooxidation were observed. They were linked to the original analyte by monitoring the disappearance of the precursor EGCG ion and the autooxidation product ions simultaneously. None of these oxidation products were observed in the plasma samples of treated mice, although they were observed to form in buffer. Metabolomics and biomarker discovery perspectives on these direct infusion MS measurements will be discussed.



P119-M

Combined Analysis of Urine Samples using NMR and LC/MSG. Zurek1, M. Godejohann2, H. Schäfer2, C. Fischer2, P. Neidig2, D. Han3, E. Lee4, M. Spraul2; 1Bruker Daltonik GmbH, Bremen, Germany, 2Bruker BioSpin GmbH, Rheinstetten, Germany, 3KBSI Seoul, Seoul, Republic of Korea, 4Korea University, Seoul, Republic of Korea.

The application of more than one analytical platform is a prerequisite to success in metabolomics in order to address large dynamic range and chemical diversity of metabolites in body fluids. The combination of LC- ESI-TOF-MS and flow injection NMR can successfully meet these analytical needs. This approach was taken in a medium scale study on human metabolism based on 1000 urine samples where we were interested in identifying influences of lifestyle (in particular smoking) on the metabolic profile as well as on methodological aspects.

Spot urine samples were automatically prepared in 96-well plates for LC-MS (1-fold dilution with water) and NMR (buffered to pH=7. The MetabolicProfiler system consists of a micrOTOF-ESI-TOF mass spectrometer cou-pled to an HPLC system and a Bruker Avance 600 MHz NMR spectrometer using flow injection technology. The oracle based sample administration and sample order man-agement system SampleTrack controlled the completely automated sample preparation and data acquisition. Data evaluation of LC/MS and NMR data was performed in an integrated approach enabling all steps required for a statisti-cal treatment of spectroscopic data.

The robustness and validity of the combined measure-ment concept on base of the integrated platform run under full automation could be demonstrated. In total 1134 urine samples were obtained from four different groups: smoker, stop-smoker, nonsmoker and people with unknown smok-ing status. In a first data screening step based on PCA, sev-eral influences typical for metabonomics based population studies were identified, e.g., smoking (nicotine and related metabolites detected by LC/MS), drug intake (drug and related metabolites detected by LC/MS and NMR), diet, age, alcohol abuse and indications on diabetes and latent inborn errors (NMR). More advanced statistical analysis and covariance analysis reveal indication on metabolic changes due to smoking status.

microfluidics

P120-T

NanoBioPrepSEQ: An Integrated, Automated, Microfluidic-Based Platform for Preparation of DNA Sequencing Samples for Capillary Array ElectrophoresisS. Jovanovich, R. Belcinski, I. Blaga, A. Boronkay, N. Burns, H. Franklin, R. McIntosh, B. Nielsen, M. Nguyen, A. Sassi, P. Sison, J. Taal; Microchip Biotechnologies, Dublin, CA, United States.

Micro- and nanofluidic technology promises to enable integrated sample-to-answer systems for many applications, improving overall performance while reducing reagent con-sumption and device size. However, the potential of such integrated devices has seldom been realized and the impact of miniaturization technologies remains largely a future vision. Here, we describe a new microfluidic platform, the NanoBioPrepSEQ station, which automates sample prepa-ration for DNA sequencing by integrating nanoliter-scale cycle sequencing and magnetic bead-based cleanup on-chip.

The NanoBioPrepSEQ uses commercial robotics to load and unload NanoBioProcessor microchips. The user places reagents and standard multiwell plates containing DNA template onto the robot and receives back Ready-to-Inject microtiter plates with samples ready for capillary array elec-trophoresis analysis, saving reagent and personnel costs. The NanoBioProcessor chip uses patent-pending, on-chip, pneumatically controlled MOV valves to mix samples and reagents, and to move beads, reagents, and wash solutions. The system is controlled by MBI’s NanoBioSoft software. Results of integrated nanoscale cycle sequencing and cleanup with readlengths over 800 bases will be presented.

The NanoBioPrepSEQ is a central part of a major col-laborative effort to develop a Microbead-based Integrated DNA Sequencer (MINDS) as a next-generation, fully integrated platform for achieving a $100,000 mammalian genome. The MINDS project and related applications of the NanoBioPrep in the development of genomic, biode-fense, forensic, and other systems will be discussed and how on-chip valves, pumps, and routers can enable many next-generation, modular, microfluidic devices for analysis of liquids. Overall strategies, designs, and results will be pre-sented to illustrate how the power of microfluidics can be leveraged to develop modular sample preparation platforms for portable and laboratory analysis.

P121-S

Enabling High-Throughput Genomics Applications in Nanoliter-Scale VolumesS. Liu-Cordero, E. Ortenberg, D. Roberts, J. Hurley, K. Papenfuss, C. Ravin, K. Munnelly; BioTrove, Inc., Woburn, MA, United States.

BioTrove has developed the OpenArray platform based on through-hole technology, a broadly applicable nanoliter fluidics technology for parallel and low-volume solution



phase reactions. OpenArray plates are coated with hydro-philic coatings on the interior of each through hole and hydrophobic coatings on the exterior of the through-holes. This enables OpenArray plates to hold solutions in the open through-holes via capillary action. The OpenArray plate consists of 3072 through-holes that can be loaded with reagents to perform individual 33 nL reactions for use in both real time PCR applications as well as endpoint genotyping applications. The unique configuration of the through-holes enables the researcher to interrogate a large number of nucleic acid samples against a large number of assays in a flexible, configurable format. By altering the number of assays or the number of samples the researcher can easily customize the OpenArray to meet their chang-ing needs. Researchers using this technology benefit from the parallelism of microarrays and the data quality of solu-tion phase reactions. Performance characteristics of the OpenArray platform will be presented and implications to genomic research will be discussed.

nucleic acid extr action /amplication

P122-M

The Effect of Ion-Pairing Reagents on the Reversed Phase Purification of DMT-off OligonucleotidesJ. R. Fisher, Jr., M. Deetz, A. Gehris, J. Maikner, M. Kinzey; Rohm and Haas Company, Spring House, PA, United States.

The use of ion-pairing reagents for reversed phase pep-tide separations has been investigated in numerous publica-tions. Reversed phase oligonucleotide separations typically employ TEAA as an ion-pairing reagent. However, other ion-pair reagents may provide better separation results. We investigated several different ion-pairing reagents using a ten micron, mono-sized, reversed phase polymeric resin, Amberchrom HPR10. DMT-off purifications of two syn-thetic 12mer DNA oligonucleotides (AAA CCT GAT GTG and GGT GGT GGT GGT) were performed, and yields and purities from the different purifications were compared in order to determine the most favorable ion-pairing conditions.

P123-T

Oligonucleotide Purification Strategies using a New High-Capacity Anion Exchange ResinM. Deetz, J. R. Fisher, Jr., A. Gehris, J. Maikner, M. Kinzey; Rohm and Haas Company, Spring House, PA, United States.

With the advent of nucleic acid silencing technologies and the need for high purity diagnostic and therapeutic oli-gonucleotides, there is a need for high-capacity chromato-graphic supports that can deliver economic purification processes. A new, 30 micron, mono-sized, polymeric resin has been recently developed that provides high resolution

and high capacity for synthetic oligonucleotides. Physical properties of this new resin will be described, including particle size uniformity, ion exchange capacity, pressure stability, and chemical stability. The purification of two synthetic 12mer DNA oligonucleotides (AAA CCT GAT GTG and GGT GGT GGT GGT) will be demonstrated, and will compare yields and purities to those from com-mercially available anion exchange resins.

other

P124-S

Wadsworth Centers’ Advanced Light Microscopy & Image Analysis Core FacilityR. W. Cole, J. N. Turner; New York State Department of Health, Wadsworth Center, Albany, NY, United States.

The Wadsworth Center is the most comprehensive state health laboratory in the country, and is dedicated to science in the pursuit of health. It fulfills its mission of protecting and promoting the health of New Yorkers through analysis, research and education combined with comprehensive clin-ical and environmental laboratory evaluation and accredi-tation programs. Our scientists study public health issues, from drug resistance to emerging infections and environ-mental toxicants as well as basic biological processes.

The Core is a resource for investigators within the Wad-sworth Center as well as those from outside institutions who require advanced light microscopic (LM) and/or image analysis as part of their research programs. Our goal is to operate a user friendly full-service Core for our user com-munity, including a comprehensive educational program. This includes the latest in imaging equipment and methods, and in image analysis software and methods. In the event that current instrumentation cannot answer the question posed by the user, we seek to develop innovative solutions. One example of this is optical projection tomography, this modality bridges the resolution gap between NMR/CAT scans and traditional LM.

No matter the specimen or the question(s) being asked we provide the technologies and assistance to get the answers. A few recent examples are: Imaging water borne contaminants to identify the organism responsible for sick-ening the patrons of a water park. Imaging motile bacteria at “near” video rates and then tracking them to investi-gate the cellular effects of an antibiotic. Visualizing in 3D, multiple biomolocules/fluorophores to investigate neuronal function in brain slices. Automatically counting cells out-growing from explants in order to map quantitative trait loci between inbred strains of mice in response to vascular endothelial growth factor. Measuring the fluidity of lipid bi-layer using FRAP to determine how lipid mobility con-trols the diffusion of small biopolymer adsorbates.



P125-M

Humoral and Cellular Immunities Induced in Mice by Enterovirus-71 Virus-Like ParticlesY. Chung, W. Chen, S. Chou, C. Chung, Y. C. Hu; National Tsing Hua University, Hsinchu, Taiwan.

Enterovirus 71 (EV71) is one of the 69 enteroviruses belonging to the picornavirus family. We have previously generated a recombinant baculovirus encoding P1 and 3CD genes for co-expressing P1 and 3CD proteins for VLP assembly in insect cells. In this study, we further charac-terized the VLP after ultracentrifugation purification, and evaluated the immunological properties of VLPs in Balb/c mice. The immunization experiments show that the VLP induced stronger anti-VP1 antibody titer and neutraliza-tion titers compared to the inactivated EV71 virus and lysed VLP. The antibodies induced by EV71 VLP were also capable of neutralizing live EV71 of different strains. Moreover, the VLP was able to induce cellular immuni-ties as judged by helper T cell proliferation assay and cyto-kine assay. These data collectively confirmed that EV71 VLP produced from baculovirus/insect cells is able to elicit humoral and cellular immunities in mice, implicating its potentials as a vaccine against EV71 infection.

P126-T

A Rapid and Sensitive Method for RNA Quality Determination on Capillary Electrophoresis SystemsA. B. Shah1, S. Karudapuram1, L. K. Joe1, J. Hauser1, A. T. Huynh1, S. Pothini1, S. Samsani1, E. S. Vennemeyer1, J. Briggs1, M. Wenz1, C. Waldron1, A. Buckelew2, C. Carver2, S. Bass2, P. Baumgartner2; 1Applied Biosystems, Foster City, CA, United States, 2Gene Logic, Inc., Gaithersburg, MD, United States.

RNA quality is directly correlated to the success of various applications, such as microarray or real time qPCR-based gene expression analyses, cDNA library construc-tion, Northern analyses, and RNAse protection assays, which utilize RNA samples from various organisms, tis-sues, cell lines, and precious biological samples. We present a novel method for examining RNA integrity and purity using capillary electrophoresis that is more cost-effective and scalable than current standard methods. This highly sensitive method uses less material, uncovers impurities fol-lowing RNA purification, and detects degradation result-ing from nuclease contamination. Total RNA and cRNA, derived from various tissues and cell lines, were stained with the dye YOYO-1 and run through a custom polymer formulation on a capillary electrophoresis platform. Using downstream analysis software, we resolved RNA species and their relative quality based on parameters such as size, profile, peak area, and peak height. Our results highlight the potential for high-throughput capillary electrophoresis as a much more discriminatory and cost-saving method in evaluating RNA quality.

pcr methods

P127-S

A Novel method to Profile miRNA Expression in Different OrganismsM. Brooks; Invitrogen Corp., Carlsbad, CA, United States.

MicroRNAs (miRNAs) are short 22-24 nucleotide RNAs that play a vital roles in regulating gene expression. These single stranded RNA molecules are incorporated into the RISC (RNA-inducing silencing complex) and regulate gene expression through various methods includ-ing translational inhibition, transcriptional cleavage and transcriptional gene silencing. Recent interest in these non-coding RNAs has been explosive because of its implication in cellular differentiation and disease genesis. Therefore, there is an urgent need to identify and validate miRNA expression in cell lines, tissues, and organs. qRT-PCR has become the standard for microarray data validation, as well as an invaluable tool for quantifying individual or subsets of miRNAs with the greatest sensitivity and accuracy. Most commercially available miRNA qRT-PCR systems employ proprietary, pre-designed miRNA-specific prim-ers for cDNA synthesis by reverse transcription. Unfor-tunately, this approach requires that the sequence of the miRNA is publicly available and a commercial qRT-PCR assay has been developed for that specific sequence, lim-iting the availability of qRT-PCR assays for many model organisms as well as recently discovered miRNAs or pro-prietary miRNAs. Here, we describe a novel, universal, and user-friendly qRT-PCR method, that was developed to measure and characterize miRNA expression in almost all organisms, starting from either total RNA and/or enriched miRNA. The NCode miRNA SYBR Green qRT-PCR protocol is based on carefully optimized polyadenylation reaction with the reverse transcriptase, SuperScript III RT, in a “universal” 1st strand cDNA synthesis reaction.. The miRNA specific amplification occurs during the PCR reac-tion where the sequence of the miRNA of interest is used as the target-specific PCR primer. Platinum SYBR qPCR Supermix combines Platinum Taq DNA polymerase with SYBR Green fluorescent dye, delivers excellent sensitivity in the quantification of target sequences. This protocol offers 7 logs of dynamic range for maximum sensitivity and single nucleotide discrimination for distinguishing between closely related miRNA families.

P128-M

SYBR Premix Ex Taq (Perfect Real Time): An Investigation of Instrument CompatibilityJ. Gibson1, T. Bio Inc.2; 1Takara Mirus Bio, Madison, WI, United States, 2Takara Bio Inc., Otsu, Shiga, Japan.

In the early 1990s the discovery that Ethidium bro-mide (EtBr) could be used to monitor PCR cycles through binding double stranded DNA (dsDNA), resulting in a fluorescence increase, ushered in a new age in PCR. By the end of the decade new detection methods using probes



(i.e. TaqMan) and instrumentation supported the launch of quantitative real time PCR (qPCR) technology. Since then many instruments and detection chemistries (FRET Hybrids, Molecular Beacons, SYBR Green and others) have been developed. Among these chemistries, the intercalat-ing chemical SYBR Green is the most similar to EtBr and widely used. It is a fluorophore that binds dsDNA in the minor groove in a non-sequence-dependent manor. Upon binding, its fluorescence increases to ~10 times that of EtBr. This detection method is simple, sensitive and relatively inexpensive to other methods currently available. Since SYBR Green detects all dsDNA, primer dimers form dur-ing PCR will also be detected and quantified. These primer dimers and non-specific amplification can be minimized by using a “hot start” polymerase which prevents reaction initiation. To help alleviate detection of primer-dimers and other non specific amplification Takara has created SYBR

Premix Ex Taq (Perfect Real Time). A convenient premix consisting of Takara’s high sensitivity-high performance Ex Taq Hot Start DNA Polymerase and SYBR Green I. Here we investigate this SYBR Green/ Ex Taq Hot Start com-bination and its compatibility with several qPCR instru-ments.

P129-T

Metalloproteinase Profiling of Cultured Cells and Isogenic Tissue in MelanomaO. Giricz, J. L. Lauer-Fields, G. B. Fields; Florida Atlantic University, Boca Raton, FL, United States.

The proteolytic activities of the ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloprotein-ase) protein families play important roles in normal and numerous pathological conditions. The ADAM family members have potential implications in the metastasis of human tumor cells via cell adhesion and protease activities. This family is characterized by the presence of both disin-tegrin and metalloproteinase domains, responsible for the adhesive and proteolytic properties, respectively. MMPs are a family of proteases responsible for the degradation of the extracellular matrix to allow cell growth and to facilitate remodeling. Under pathological conditions these proteases are involved in many diverse processes from tumor cell migration to cartilage destruction in rheumatoid arthritis.

The gene expression levels of eight ADAMs, previ-ously described to possess proteolytic activities; and seven MMPs, previously described to have significant roles in the remodeling of the extracellular matrix during the meta-static process, were analyzed by Real Time PCR using an ABI SDS Prism 7000. RNA was isolated from multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic normal tissue and tumor samples. This method allowed for detected changes in mRNA expression of the individual metalloproteainase genes to be compared between normal and metastatic states, and also between tissue and cultured cells.

Based on the preliminary results, there are substan-tial differences in the level of ADAM and MMP mRNA expression between tissue and cell lines. In general, the level of expression is several fold higher in cultured cells compared to the isogenic tissue they are derived from. We

are currently performing in situ fluorogenic enzyme assays on cultured cells to determine if the elevated expression in accompanied by enhanced proteolytic activity.

P130-S

Development and Evaluation of an Absolute qPCR Technique for Methicillin-Resistant Staphylococcus aureus (MRSA) in Ventilator Associated Pneumonia (VAP) by using Mini-bronchoalveolar Lavage (mini-BAL) SpecimensX. Wang1, L. Goodwin1, C. C. Ginnochio2, S. Condon2, P. Francois3, D. Ost4,5; 1Molecular Genetics/Core Facility, General Clinical Research Center, Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health System, Manhasset, NY, United States, 2Microbiology, Virology and Molecular Diagnostics Laboratory, North Shore-Long Island Jewish Health System, Lake Success, NY, United States, 3University Hospital of Geneva, CH-1211 Geneva 14, Switzerland, 45Interventional Pulmonology, North Shore University Hospital, North Shore-Long Island Jewish Health System. (Currently, Division of Pulmonary and Critical Care, New York University School of Medicine.), Manhasset, NY, United States, 5This study was supported 100% by the North Shore LIJ Health System General Clinical Research Center, Grant # M01PRO18535., Manhasset, NY, United States.

An absolute qPCR technique was developed and evalu-ated for detection and identification of methicillin-resis-tant Staphylococcus aureus (MRSA) directly in mini-bron-choalveolar lavage (mini-BAL) clinical specimens from patients with ventilator-associated pneumonia (VAP). This absolute qPCR technique measures simultaneously three target genes: mecA gene, conferring methicillin resistance that is common to both Staphylococcus aureus and Staphy-lococcus epidermis; the femA-SA gene from S. aureus; and the femA-SE gene from S. epidermis. A mecA gene standard curve from serial dilution of plasmids containing known amounts of mecA gene fragments was incorporated in each plate. This approach not only discriminates of the origin of the measured mecA signal but also provides the absolute copy number of mecA gene existing in MRSA, which is very informative for choosing the proper antibiotic therapy and monitoring the efficacy of the therapy for clinician. PCR inhibitor in two nucleic acid extracts was detected by SPUD qPCR and excluded from this study. Absolute MRSA qPCR was able to distinguish MRSA from methi-cillin-resistance Staphylococcus epidermis (MRSE), methi-cillin-sensitive Staphylococcus aureus (MSSA), methicillin-sensitive Staphylococcus epidermis (MSSE) based on the amplification profiles of three genes; and could detect the MecA gene down to a copy number of 3 with a high degree of linear correlation (R2 = 0.999) in all 12 different runs. Using quantitative culture results as a gold standard for MRSA diagnosis, the sensitivity and specificity of this assay were 90.48% and 94.47% respectively. When we used >100 copies /ml mecA gene combined with positive femA-SA as cutoff for MRSA diagnosis, the positive predictive value increases from 82.61% to 94.12% with the negative pre-dictive value decreases from 97.26% to 96.20%. Absolute



MRSA qPCR is the first assay that provides fast, quantita-tive, highly sensitive, and specific for detection of MRSA in mini-BAL clinical specimens from VAP patients.

peptide synthesis

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Performance Evaluation of a New 6-Channel, Small-Scale Peptide SynthesizerP. Campbell1, J. Park2, H. Patel2, T. Racioppo2, J. Blais2, B. Hensley2, N. P. Ambulos, Jr.1; 1University of Maryland School of Medicine, Baltimore, MD, United States, 2Protein Technologies, Inc., Tucson, AZ, United States.

Protein Technologies has developed a new parallel pep-tide synthesizer able to perform efficient synthesis at scales from 10 to 350 µmoles. A bench-top model, the Prelude incorporates several desirable features, including extra sol-vent and amino acid bottle positions, ‘single-shot option’ for specialty amino acids, and an effective mechanism for improved solvent washes. Our objective was to field test the performance at the lower scale range of this instru-ment, evaluate the quality peptides with diverse sequences, including modified amino acids and derivitization, and to compare the results with those obtained from parallel syn-theses on other instruments.

We synthesized approx. 80 peptides. The sequences were anionic, cationic and hydrophobic, linear and branched. Some contained phosphorylated amino acids, and some of the peptides were derivitized with biotin or fluorescein. Three quarters of the peptides had a crude purity greater than 50%, many of those exhibited purities that were bet-ter than 80%. Seven extra amino acid reservoirs permitted unattended synthesis (no manual additions) of peptides containing phosphoserine and phosphotyrosine as well as several pseudoprolines. Derivitizations also proceeded automatically, as did hydrazinolysis of [(4,4-dimethyl-2,6-dioxycyclohex-1-ylidene)ethyl] (Dde) protective groups. High performance liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry will illustrate the conclusions.

P132-T

A Simple Protocol of Preparing N-myristoylated PeptidesG. Jayathilaka, S. Lateef, S. Gupta, S. Krishnanchettiar, B. Lee; UIC, Chicago, IL, United States.

Protein N-myristoylation is an early event in acylpro-tein biosynthesis. There are many known N-myristoyl-proteins such as cAMP-dependent kinase, calcineurin B, p60src, cytochrome b5 reductase, p56LSTRA/TCK, MuLV gag, M-PMV gag, BLV gag, MMTV gag, FeLV gag, BaEV gag, HTLV gag, HIV gag, HIV 3’-orf, Hepatitis B Virus pre-S1, Polyoma Virus VP2, SV40 Virus VP2, Poliovirus VP4, Bovine Enterovirus VP4, bovine brain G0 α-sub-unit. A number of these N-myristoylated proteins are pro-

tein kinases or phosphatases that have important roles in modulating cellular metabolism. A variety of observations suggest that N-myristoylation may participate in the jux-tapositioning of these acylproteins and other components of cellular regulatory circuits. This might occur either by directing these proteins to particular cellular membranes, or by permitting them to interact with other polypeptides that reside in either the cytoplasm or membranes. N-myris-toylpeptides are being used to investigate the roles of the N-myristoylation. We present here a simple procedure of making the N- myristoylated peptides in this Laboratory. Briefly, the fully protected peptides (~0.05 mmoles) on res-ins (~0.1 g) using 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry are de-Fmoced by 20% piperidine in dimeth-ylformamide (DMF). After washed resins with DMF and dichloromathane (DCM), resins are mixed with 5 mL of 50% chloroform in DMF containing ~5 g myristic anhydride and kept at 65°C for 1.5 h. After that, resins are washed several times with the hot 50:50 chloroform:DMF solution. Finally N-myristoylpeptides are cleaved of resins and deprotected by a standard cleaving procedure. Compare to the procedure used by Dr. Glaser which uses pyridine at 23°C for overnight with mixing, our method is faster and do not use pyridine.

P133-S

An Improved Protocol of Coupling Synthetic Peptides to KLH for Antibody Production Using DMFS. Lateef, S. Gupta, G. Jayathilaka, S. Krishnanchettiar, B. Lee; UIC, Chicago, IL, United States.

Keyhole limpet hemocyanin (KLH) conjugated pep-tides are routinely used as antigens to generate antibody. In the traditional method, peptides are dissolved in phosphate-buffer saline (PBS) and then mixed with 3-maleimidoben-zoic acid N-hydroxysuccinimide ester (MBS) conjugated KLH to make KLH conjugated peptides. If the peptide is not soluble in PBS, it can be dissolved in 6 M guanidine-HCl/0.01 M phosphate buffer, pH 7.0. However, there are peptides which are insoluble in both buffers. We present here a procedure of making the KLH conjugated peptides using dimethylformamide (DMF) instead of PBS or 6 M guanidine-HCl to increase solubilities of peptides. Addi-tionally, the last desalt step in the traditional method is eliminated. Briefly, 5 mg of peptide is dissolved in 100 µL of DMF. This solution is added to 1 ml of purified KLH-MBS slowly. The pH of the solution is adjusted to ~7.0 immediately by adding either 2 N NaOH or 0.5 N HCl. The solution is then stirred or rotated for 3 h or overnight at 4°C. Many peptides which do not dissolve in 6 M gua-nidine-HCl will be soluble in DMF. Some peptide solu-tions may give cloudy or gelly appearance, nevertheless, peptides do react with KLH to some extend and solutions will become clear. The reaction can be kept longer for such peptides. Also, some peptide-KLHs do form precipitates which can be left in the final solution. Finally, 2 ml of 0.1 M ammonium bicarbonate is added to the peptide/KLH solution, and the solution is lyophilized. Compare to the traditional method, our method is faster and peptides with



solubility problems in PBS or 6 M guanidine-HCl can be used.

P134-M

Solid Phase Synthesis of C-Terminus Fluorescent Peptide: Comparison of Fmoc-Lys(5-FAM)-Resin and Fmoc-Lys[5-FAM(Trt)]-ResinX. Tong, W. Fu, L. Sheng; AnaSpec Inc., San Jose, CA, United States.

Many FRET (Fluorescent Resonance Energy Transfer) peptides require a C-terminus fluorescent label. In order to facilitate the synthesis of C-terminus fluorescent pep-tides, we prepared and compared two kinds of resins, Fmoc-Lys(5-FAM)-resin (I) and Fmoc-Lys[5-FAM(trt)]-resin (II).1 Resin (II) has a phenolic hydroxyl group protected with trityl group. The reason for introducing the protect-ing group was supposedly to prevent the phenolic hydroxyl groups from reacting with the activated amino acids during peptide synthesis.

In this report, a fluorescent peptide [EREQTVDLS-VKRPRTGRKKRRQ-RRRK(5-FAM)-NH2] was synthe-sized using each of these resins. Syntheses were carried out under similar standard conditions. The peptides obtained showed no significant difference in purity. These results showed that resin (I) is also suitable for synthesis of C-ter-minus fluorescent peptide.

reference

1. Fischer R, Mader O, Jung G, Brock R. Extending the applica-bility of carboxyfluorescein in solid-phase synthesis. Bioconju-gate Chem 2003;14:653–660.

protein analysis

P135-T

Structural Characterization of a Recombinant Monoclonal Antibody by Electrospray Ion-Mobility Time-of-Flight Mass SpectrometryW. Chen, P. Olivova, A. B. Chakraborty, M. Gilar, J. C. Gebler; Waters Corporation, Milford, MA, United States.

Recombinant monoclonal antibodies (MAb) com-prise a significant proportion of biopharmaceuticals used in diagnostic and therapeutic applications. Although the general structural features of monoclonal antibody such as disulfide bond patterns have been known for decades, the intrinsic heterogeneity of such molecules has imposed the needs for thorough analytical characterizations so the structural details of each pharmaceutical MAb are deter-mined, and safe, effective, and reproducible products can be generated.

The analysis of intact MAb molecules is challenging due to their high molecular mass, hydrophobic nature, and presence of sugar moieties. Conversely, the analysis of intact protein is also advantageous because it reduces dra-matically the time for sample preparation and data inter-

pretation compared with peptide mapping and sequencing. Furthermore, it also minimizes the chance of introducing putative modifications, which are often observed during peptide mapping.

The aim of this study is to perform structural charac-terization of a recombinant monoclonal antibody (IgG1) by a newly developed electrospray ion-mobility time-of-flight mass spectrometer (ESI-IM-TOF MS) using a top-down approach. In this approach, the molecular masses of the intact and the deglycosylated antibodies as well as the light and heavy chains of the antibody were measured by direct infusion to MS. The mass difference between intact and the deglycosylated antibody establishes the glycosylation profile of the antibody. For the reduced antibody, the ligh and heavy chains are separated in the gas phase by the ion mobility functionality of the instrument. Thus, the masses of light and heavy chains are accurately measured to yield further evidence for other post-translational modifications. Furthermore, the fragmentation of the intact light chain subunit and subsequent separation of fragment ions by ion mobility allows us to quickly obtain partial sequence of the light chain. All of the results demonstrate that ESI-IM-TOF-MS is a superior tool to characterize MAbs and other complex protein pharmaceuticals.

P136-S

Traceless, Fluorescent Monitoring of Proteolytic DigestionH. Y. Choi1, D. A. Veal1, A. Crawford2, G. B. Scott2, P. H. Karuso1,3; 1FLUOROtechnics, Sydney, Australia, 2Sigma Aldrich, St Louis, MO, United States, 3CBMS, Macquarie University, Australia.

The objective is to develop a simple, real-time method for monitoring protease activity.

Proteolytic digestion prior to identification by MS is pivotal to contemporary proteomics. Proteolytic diges-tions are taken between 1 and 18 h to complete, depending on the protein, enzyme activity and reaction conditions. Ensuring that digestion is stopped at the optimal time is important as failure, partial digestion or over-digestion can waste MS-time and make results difficult to interpret. Monitoring proteolysis is also important in areas ranging from digestibility studies of foods to removal of tags or fusions from recombinant proteins. Standard techniques for monitoring proteolysis (e.g., HPLC, circular dichroism, SDS-PAGE) are slow, may require expensive instrumenta-tion, can be inaccurate and/or are unsuitable for real-time monitoring.

Epicocconone yields an intensely red-fluorescent prod-uct on reversible binding to lysine, arginine and histidine residues in the hydrophobic environments around proteins. This feature provides a novel approach for staining and quantification of proteins and peptides in solutions, gels, blots, and cells that is compatible with downstream pro-cesses such as HPLC, MS, Edman degradation and West-ern blotting.

LavaDigest is a new product that is based on epicocco-none which measures the hydrophobicity loss as a decrease in fluorescence as a protein or proteome is digested. This provides a simple, approach to monitoring proteolytic



activity. LavaDigest appears suitable for most proteins and proteases tested; it does not interfere with proteolytic activ-ity, is compatible with down-stream proteomic analysis and can be used to derive kinetic parameters of proteolytic activity. LavaDigest has been used to compare the rates of degradation of different proteins, by a variety of proteases under different conditions (pH, surfactants, temperature, etc). The assay is unique in that it can be used to tracelessly follow the activity of proteases on native substrates.

P137-M

Rapid Removal of Detergents from Protein Solutions using Polymeric Reversed Phase ResinsA. Gehris, J. R. Fisher, Jr., J. Maikner; Rohm and Haas Company, Spring House, PA, United States.

Detergents are commonly used in hydrophobic cell membrane protein purification and viral removal/inactiva-tion. These detergents must eventually be removed during the purification process. A rapid detergent removal protocol using short chromatographic columns has been developed. Polystyrene divinylbenzene polymers, packed in chroma-tography columns, were employed for the removal of non-ionic, zwitterionic, and anionic detergents from protein solutions. The results demonstrate that this resin exhibited high capacity and was effective in removing a wide range of detergents at high linear velocities (>600cm/hr) with mini-mal loss in protein yield.

P138-T

Microwave-Assisted Protein HydrolysisE. S. Ingle, J. R. Lill; Genentech, South San Francisco, CA, United States.

Introduction: Protein hydrolysis is the process by which peptide bonds are broken under acidic conditions at high temperatures, generating free amino acid residues. The resulting hydrolysates can then be analyzed by amino acid analysis to determine the amino acid composition and con-centration of proteins. The hydrolysis procedure is the rate-limiting step for amino acid analysis. Microwave-assisted protein hydrolysis offers the potential to reduce hydrolysis time significantly.

Previous microwave studies indicate that high-tempera-ture/short-time exposure can be applied to the hydrolysis of proteins for amino acid analysis. Currently for high-throughput protein hydrolysis, samples undergo vapor-phase hydrolysis in 6 N hydrochloric acid with phenol for either approximately 24 hours at 110°C or for 1 h at 150°C. Our studies evaluated various protein hydrolysis methods by comparing microwave irradiation to traditional hydro-lysis conditions.

Materials and Methods: For the microwave-assisted hydrolysis studies, protein standards were hydrolyzed by microwave irradiation for 10 minutes at 175°C using the CEM Discover System (CEM Corp., Matthews NC). Pro-tein standards were also hydrolyzed at either 110°C for 24 h or 150°C for 1 h in a Water Pico•Tag Workstation (Waters, Corp, Milford, MA). The hydrolysates were then analyzed

using the Hitachi L-8800 Amino Acid Analyzer (Hitachi High Technologies America, San Jose, CA) .

Results: Protein standards hydrolyzed by microwave irradiation had comparable chromatographic profiles and amino acid recoveries to proteins that underwent tradi-tional hydrolysis at 110°C for approximately 24 h or at 150°C for 1 h.

Conclusions: Protein hydrolysis by microwave radiation offered a significant time savings (10 minutes compared to up to 24 h), allowing for faster sample analysis. Microwave irradiation has been demonstrated as an acceptable alterna-tive to the traditional protein hydrolysis conditions.

protein arr ays

P139-S

Antibody Microarray Analysis of Inflammatory Mediator Release by Human Leukemia T cells and Human Non-Small Cell Lung Cancer CellsB. Garcia1, A. Hargrave2, E. Hommema1, G. Kilmer1, A. Morgan1, J. Narahari1, B. Webb1, R. Wiese1; 1ThermoFisher Scientific, Rockford, IL, United States, 2University of Chicago, Chicago, IL, United States.

Cytokines and chemokines are responsible for regulat-ing inflammation and the immune response. Cytokine and chemokine release is typically quantitated by ELISA or Western blot analysis. In order to expedite the analysis of samples for multiple cytokines/chemokines, we have devel-oped slide-based ExcelArray antibody sandwich microar-rays. Each slide consists of 16 subarrays (wells) printed with 12 specific antibodies in triplicate and positive and nega-tive control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the “sandwich” ELISA, in which an analyte protein is “sandwiched” between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked DyLight 649 dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. The Jurkat cell line was used as a model for human T cell leukemia and the A549 cell line was used as model for human non-small cell lung cancer in our experiments. In order to evoke a cytokine/chemokine response, cells were stimulated with Tumor Necrosis Factor alpha (TNFα), Phorbol-12-myristate-13-acetate (PMA, TPA) and phy-tohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angio-genesis and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-α, and MIP-1α, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell culture supernatants for the profiling of cellular inflammatory mediator release.



P140-M

Specificity Analysis of Therapeutic Antibodies using Protein BiochipsA. Lueking1, C. Gutjahr1, V. Gruß1, K. Schulte1, S. Müllner1, R. Garretson2, J. Beator1; 1Protagen AG, Dortmund, Germany, 2Protagen Inc., Chester, NJ, United States.

Protein Microarrays enable the analysis of multiple binding events in parallel. Based on patented UNIclone technology, we have established a reproducible protein biochip platform to determine specific binding profiles to multiple targets in a quantitative way. The UNIchip pro-tein biochips provide a novel and unique tool for antibody characterization and ranking. Potential off-target activi-ties can be detected by establishing a quantitative binding profile of an antibody to 400 different, unrelated targets. Together with on-chip determination of sensitivity, linear-ity and dynamic range the quantitative fingerprint enables a unique performance ranking and specificity analysis of antibodies. We have analyzed the performance of three block-buster biotherapeutics directed against TNF-alpha. The biotherapeutics show a clear differentiation both with regard to their binding profile to unrelated proteins as well as their binding affinity to the cognate target on the bio-chip. The quantitative fingerprint also offers a novel strat-egy for antibody pair selection in ELISA development.

P141-T

A New Array Format for Protein Kinase Substrate DeterminationH. Gausepohl, O. Brandt; INTAVIS Bioanalytical Instruments AG, Koeln, Germany.

Peptide arrays are useful tools to characterize antibod-ies, enzyme substrates or sequence specificities of interac-tion partners with given peptide sequences (e.g., SH2, SH3, MH2 and other domains). Here we present a new method1 that allows production of hundreds of identical peptide arrays from a single synthesis run on modified, individual cellulose-disks. The disks are dissolved in the acid cleav-age-mixture after synthesis and the resulting solutions of peptide-cellulose-conjugates are then spotted onto multiple slides by conventional spotting techniques. As application example we show results obtained with arrays of kinase substrate libraries and various consensus sequences of known kinase targets. These arrays can be used with dif-ferent detection methods to profile known and unknown kinases for their substrate specificity.

The new arrays are derived from the the well known SPOT method2 but offer several major improvements: A smaller volume of sample (only 100 µL) is needed for incu-bation, and a high number of identical copies of the arrays enables large scale, parallel screening experiments. The cost of an individual array is considerably lower than that of a SPOT membrane. Unlike DNA hybridization, protein-pro-tein interactions frequently suffer from low binding affini-ties. The new cellulose substrate with peptides linked to it generates a three dimensional scaffold on the array support with a peptide loading exceeding that of a monolayer by a factor of 100. The high peptide density of the spots should

be advantageous to identify protein-interaction sites, even if their binding constants are low.

references

1. Beutling, U., et al. (2005) Kenes International, Geneva, pp. 152–153.

2. Frank, R. Tetrahedron (1992) 48, 9217.

P142-S

Novel Uses of Microarrays in Detecting Gene SilencingE. T. Jordan, C. Karlak, T. Rubio, L. Ugozolli, J. Wibbenmeyer; Bio-Rad Laboratories, Hercules, CA, United States.

DNA microarrays allow many simultaneous parallel measurements; and transcriptional profiling has provided scientists with a wealth of information. The same technol-ogy can be used to build protein arrays which hold simi-lar promise. We describe here an approach using protein arrays to screen cells for gene knockdowns. Small interfer-ing RNAs (siRNA) can be used to degrade mRNA lev-els of a specific gene, thereby reducing the corresponding protein levels within cells. The use of siRNAs has many implications in functional genomics and proteomics as well as therapeutics, as researchers use this tool to identify pro-teins involved in specific pathways. We demonstrate the use of microarrays as a screening method to identify cells that have been treated successfully with a beta-actin siRNA.

We have silenced the β-actin gene in HeLa cells using siLentMer Dicer-Substrate siRNA Duplexes, and con-firmed the knockdown with a combination of 2-D gel analysis, western blotting and quantitative PCR (qPCR). An increase in the level of phosphorylated cofilin was also detected (Liu, N., et al. 2006 in press).

We now demonstrate the use of reverse phase protein microarrays to screen for these proteins. These arrays enable higher throughput than traditional protein detection, and add multiplexing capabilities. Arrays were produced on the benchtop with the BioOdyssey Calligrapher miniarrayer. Antibodies against β-actin and phospho-cofilin were tested for specificity by western blots. In one experiment, arrays were processed to monitor the concentration of β-actin in cells, with a standard curve of purified human actin printed on the grids. In another experiment, changes in phosphorylation levels of cofilin with an antibody specific for phospho-cofilin were detected. We also demonstrate that these printed arrays can be screened using antibodies either singly or in pairs. Finally, the microarray results were validated using qPCR.



P143-M

Microarray-Generated Phosphopeptide Selectivity Profiles for Fluorescent Conjugates of an Artificial Phosphomonoester-Binding Small Molecule; Implications for the Global Enrichment and Detection of Phosphopeptides and PhosphoproteinsW. F. Patton1, J. Zerweck2, H. Xie1, S. Kramer1, D. Sissors1, M. Schutkowski2; 1Perkin-Elmer Life and Analytical Sciences, Waltham, MA, United States, 2JPT Peptide Technologies GmbH, Berlin, Germany.

Reliable identification and characterization of protein kinase substrates is of paramount importance to decipher-ing cellular signaling pathways. Previously, an artificial phosphomonoester-binding molecule, 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato Zn2 complex (a.k.a. Phos-tag) was shown to bind to phenylphosphate under physiological conditions with a Kd of 25 nM. A fundamen-tal challenge in chemical biology is efficiently performing comprehensive profiling of the activity of artificial chemi-cal structures, such as the Phos-tag molecule, against the hundreds or even thousands of biological targets they could potentially interact with. A peptide microarray-based phosphorylation site screening assay was thus performed to determine the binding selectivity of fluorophore-con-jugated Phos-tag molecule, based upon direct detection of binding through readout of fluorescent signal generated by the reporter group. Epitope mapping was performed on a comprehensive list of phosphorylation sites known to be expressed in the human proteome, including serine, threo-nine and tyrosine phosphorylated residues. The collected data was evaluated using a combination of different in-house software packages resulting in scores for each amino acid residue relative to the phosphorylation site. The study demonstrates that fluorophore-conjugated Phos-tag exhib-its very broad substrate specificity and does not appear to depend upon a specific sequence surrounding the targeted phosphorylation site. This relatively low degree of sequence restriction at the phosphorylation recognition site suggests that Phos-tag is broadly suitable for universal enrichment and detection of phosphorylation sites on phosphoproteins and phosphopeptides. The fluorescent Phos-tag technology is easy to perform, cost effective and should allow rapid large-scale screening of protein and peptide phosphoryla-tion using a variety of solid-phase assay formats.

protein sequencing techniques

P144-T

Top Down Sequence Analysis of Intact Protein Species Using a Novel Travelling Wave Ion Mobility Device Coupled With a Time-of-Flight Mass SpectrometerT. McKenna, C. J. Hughes, S. Pringle, K. Giles, J. I. Langridge; Waters Corporation (MS Technologies Centre), Manchester, United Kingdom.

In this presentation we will demonstrate the utility of Ion Mobility Spectrometry in the top down sequencing of

proteins. These data were acquired using a novel Quad-rupole/TWIMS/oa-Tof mass spectrometer, operated with a nanoelectrospray ion source. The TWIMS is a stacked-ring ion guide, operated at elevated pressure, with oppo-site phases of an rf voltage applied to adjacent plates to provide radial ion confinement. A continual sequence of dc pulses is superimposed on the confining rf to provide ‘waves’ which propel ions through the gas. Protein species were ionised and the resulting ions separated based upon their ion mobility, or collision cross section, through the TWIMS device and subsequently mass analysed using the oa-TOF analyser.

We have investigated the use of the hybrid ion mobility/time-of-flight system for the top down analysis of proteins and compared these data with the data that can be obtained without an IMS separation. We have fragmented different charge states of the same protein and, by the post analy-sis selection of different regions of m/z vs drift time plots, produced spectral information which contains fragment ions of similar charge states. These selected regions can reveal species that are hidden in the non mobility experi-ment and these specific charge state spectra can be sub-sequently deconvoluted to produce sequence information. Typical increases in sequence coverage of around 20–25% are observed with a mobility enabled acquisition compared to a non mobility experiment.

P145-S

Interactive and Automated Top-Down Analysis of Sequencing Data of Intact ProteinsD. Suckau1, L. Vorwerg1, A. Resemann1, M. Witt1, M. Easterling2; 1Bruker Daltonik GmbH, Bremen, Germany, 2Bruker Daltonics, Billerica, MA, United States.

In a Top-Down workflow (ECD, ETD, ISD) the whole protein molecule is subjected to fragmentation and pro-teins are not digested (as in the Bottom-Up approach). This allows the detection of signal peptides, modifications, sequence variations and mutations. Top-Down analyses can be done using FTMS/ECD/ESI or MALDI-ISD spectra.

For the analysis of Top-Down datasets f lexible algo-rithms are required that allow the creation of sequence tags based on peak mass differences and several ways to process the sequence tags in a subsequent step. Sequence tags can be created that can include various modifications, user-defined mass range, inclusion of selected peaks and exclusion of peaks matched by accepted tags. The resulting sequence tags are scored and can be used to perform MS-BLAST homology searches or Mascot sequence tag searches to identify unknown proteins. An alternative workflow that was developed involves a known protein sequence and the identification of protein modifications or mutations. Using these algorithms it is possible to automatically suggest the signal peptide structure from the mass offset between the experimental tag and the theoretical value that is based on the protein sequence database entry, modifications or a mutations.

We used the algorithms that are included in the BioTo-ols 3.1 software package to analyze reISD-MALDI-TOF and ECD-FTICR spectra from undigested proteins in the molecular weight range 6-70 kDa and could automatically



detect, e.g., the length of the pre-pro peptide of bovine serum albumin (67 kDa). Other examples involved pro-teins with N/D and Q/E ambiguities in their database record such as carbonic anhydrase. An interesting field of application is the localization of protein phosphorylation sites based on the undigested protein as in the top-down analysis suppression of the phosphorylated peptides is not observed.

proteomics

P146-M

A Fast, Easy, Accurate Method for Protein QuantitationN. Ahnert, J. Dallwig, T. Goodman, G. A. Thomas, M. Beaudet; Molecular Probes, Invitrogen Detection Technologies, Eugene, OR, United States.

Standard protein quantitation is an involved process, often including serial dilution of standards and plotting of sample values on a curve of values obtained from the dilution series. Through integration of fluorescent reagents and a low cost fluorescent instrument programmed with a curve fitting algorithm, we have developed a fast and accu-rate method of protein quantitation that eliminates both calibration standard preparation and manual calculation. In the Quant-iT Protein Assay for the Qubit fluorometer, a fluorescent reagent is mixed with a dilution buffer, and the mixture is added to only three premixed calibration standards and to all samples. Instrument readings of the three standards are automatically fit to a sigmoidal curve, allowing concentration of the sample in µg/mL to be the only output. The assay is accurate for initial sample con-centrations from 12.5 µg/mL to 5 mg/mL and exhibits low protein-to-protein variation compared to other protein quantitation methods. The assay is performed at room tem-perature, and the signal is stable for three hours. Common contaminants, such as reducing agents, salts, free nucleo-tides, amino acids, solvents, buffers, and DNA or RNA, but not detergents, are well tolerated in the assay. Samples in standard protein 1D, but not 2D, gel loading buffer may be diluted and then quantitated in the assay. The entire assay takes about 30 min to perform.

P147-T

Differential Proteome Analysis of Primary and Metastatic Melanoma Using 2DE and LC-MS/MSM. Al-Ghoul, T. Brueck, J. Lauer-Fields, V. Asirvatham, R. Kerr, G. B. Fields; Florida Atlantic University, Boca Raton, FL, United States.

Melanoma causes the highest rate of skin cancer related fatalities. If detected and treated early, melanoma is mostly curable. Unfortunately, once metastasis has occurred mela-noma is often fatal. The biochemical pathways involved in the development from primary to metastatic melanoma is an area under intense investigation. A high-throughput

proteomics approach has been applied to better understand the processes that underlie tumor formation and progres-sion. Analyses of global melanoma proteome expression patterns are compared between the primary melanoma cell line WM-115 and the metastatic melanoma cell line WM-266-4, where both cell lines derived from the same patient. Total cell lysates were separated by two dimensional-gel electrophoresis (2DE). After protein differential expression analysis, protein spots of interest were excised, digested with trypsin and analyzed by LC-MS/MS. The mass data were searched against an in-house NCBInr database using the Mascot search engine for protein identification. Com-paring the spots between gels, 470 spots were matched for both gels, while 109 spots were up-regulated and 15 spots down-regulated in metastatic melanoma. We identified proteins involved in tumor progression, metabolism, signal transduction, DNA binding, as well as structural and heat shock proteins. This study establishes a step forward in the development of the metstatic melanoma protein database, the understanding of the chemical pathways that lead from primary to metastatic melanoma, and identifying new tar-gets for inhibitor development.

P148-S

A Sensitive and Simple Procedure for Staining Proteins on Either Nitrocellulose or PVDF Membranes Based on the Fluorophore EpicoccononeM. S. Ball1, C. Steller1,2, R. Sandalnha3, J. Hong4, R. Simpson4, P. Karuso1,5; 1Fluorotechnics Pty Ltd, Sydney, Australia, 2Fachhochschule Giessen-Friedberg, University of Applied Sciences, Department KMUB, Fachhochschule Giessen-Friedberg, Germany, 3Australian Proteome Analysis Facility, Sydney, Australia, 4The Ludwig Institute, Melbourne, Australia, 5Department of Chemistry & Biomolecular Sciences – Macquarie University, Sydney, Australia.

Epicocconone, a naturally occurring fluorophore that alters its fluorescent character upon binding to proteins is the basis for a number of new labeling technologies ranging from liquid protein quantitation to gel stains and cell stains. LavaPurple is a highly sensitive total protein gel stain that may be used as a stain for nitrocellulose and PVDF blots. The high sensitivity of the stain, coupled with the unique reversible covalent binding of epicocconone, make it an ideal blot stain as it is easily removed by pH change thus not interfering with downstream immuno-staining, mass spectrometry or Edman sequencing.

Aim: This study measures the sensitivity of LavaPurple as a blot stain on both nitrocellulose and PVDF membranes, as well as the compatibility of the stain with immuno-stain-ing.

Method: a dilution series of Low molecular weight SDS-PAGE calibration markers (GE Healthcare) were separated by electrophoresis and electro-blotted onto either nitrocel-lulose or PVDF. The blots were then stained with LavaPur-ple and image analysis performed to determine sensitivity and linearity of the stain. Cell lysate from ovarian cancer cells and rat fibroblasts were separated by 2D GE and blot-ted onto both nitrocellulose and PVDF membranes before



staining with LavaPurple. The blots were then used for immuno-staining and the efficiency of visualization com-pared with and without total protein pre-staining.

Results: LavaPurple was able to visualise proteins on blots at less than 250pg/band loaded on a 1D gel and did not inhibit subsequent antibody binding to target proteins on either 1D or 2D blots. Furthermore the stain could be easily removed from the blot for subsequent staining with other fluorophores.

Conclusions: LavaPurple is a highly sensitive stain that is suitable for total protein visualization on blots either for spot removal or prior to immuno-staining.

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Proteomics Sample Preparation Using Magnetic Bead Based TechnologiesS. Bergholtz, E. Breivold, D. Tran, E. Ragnhildstveit, O. Brekke, K. Evensen, G. Fonnum, M. Bosnes, D. Gillooly; Invitrogen Dynal, Oslo, Norway.

We have developed a range of magnetic beads for pro-tein and/or peptide sample isolation and fractionation that can be utilised in proteomics strategies and workflows.

A significant challenge in proteomics today is over-coming the dynamic range of protein abundance. High throughput proteomics requires efficient methods to ana-lyze complex protein mixtures. It is often necessary to reduce sample complexity for many proteomics strategies. The use of magnetic beads for sample preparation enables protocols to be automated and throughput to be increased. The kinetics of bead-based sample preparation is very effi-cient and washing can be done thoroughly. In addition, preparation is flexible regarding sample and buffer volumes. Consequently, low abundant proteins can be concentrated from large sample volumes.

We have developed protein purification methods based on the use of magnetic bead technology. These methods are used for the fractionation of complex protein samples and for isolation of proteins or peptides.

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Utilization of ABRF sPRG Protein Standard for Developing Optimized Experimental Strategies for ETD Based Protein IdentificationR. G. Biringer, Z. Hao, A. F. R. Hühmer; Thermo Fisher Scientific, San Jose, CA, United States.

Conventional methodology of protein mass spectrom-etry using collision induced dissociation (CID) has been widely adopted for confident identification and character-ization of proteins in recent years. However, CID-based protein analysis works best with relatively short peptides and many important post translational modifications are not preserved during CID-based analyses. A new frag-mentation method, electron transfer dissociation (ETD), has been developed recently and introduced to linear ion trap mass spectrometer. In ETD, peptide backbone is frag-mented along pathways that are analogous to those observed in ECD. ETD has been reported to not only favor multiply

charged, relatively large peptides, but also preserve post translational modifications. Thus, ETD is drawing more and more attention as mass spectrometry based methods for protein identification and analysis of post-translational modifications. However, current experimental strategies, which perfectly match CID-based analysis, are not opti-mized for ETD. This lack of efficient experimental strate-gies that enhance ETD-based analysis could significantly limit the benefits which ETD can provide for the analysis of proteins.

The purpose of this study is to optimize experimen-tal strategies for the comprehensive analysis of the ABRF sPRG Protein Standard and to compare sensitivity of CID and ETD activation methods using various concentrations of this standard. Protease digested ABRF sPRG 49 protein standard mixture was analyzed on a Finnigan LTQXL with ETD employing the combination of CID and ETD acti-vation types. Comparison of data generated by ETD and CID indicates complementarity of amino acid sequence coverages in the resultant datasets. Many peptides only show strong fragmentation patterns by either one or the other method. However, improved protein sequence cov-erage was enabled by alternating between CID and ETD activation types. Increased protein sequence coverage not only allowed a more thorough characterization of the 49 proteins in the standard, but also reduced the number of false positive protein hits.

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A Complimentary Proteomics Study to Identify Genes Regulated by a Translational Regulator and an RNA-Binding Protein in Pseudomonas aeruginosaA. Brencic1, J. C. Silva2, C. Dorschel2, S. Lory1; 1Harvard Medical School, Boston, MA, United States, 2Waters Corporation, Milford, MA, United States.

P. aeruginosa is an opportunistic human pathogen and a major cause of death in patients with cystic fibro-sis. Recently, an elaborate regulatory network has been discovered that regulates transition between two different virulent modes of the bacterium, one that causes an acute and another that causes chronic infection. In response to unknown environmental signals this network modulates activity of a single protein, RsmA. RsmA is a translational regulator and an RNA binding protein. The objective of this work is to identify direct mRNA targets of RsmA and to elucidate the mechanisms underlying RsmA-mediated regulation.

Using a data-independent, label-free LC-MS method, protein expression patterns were monitored in wt, rsmA mutant, and rsmYZ double mutant (analogous to a strain overexpressing RsmA) cultures of P. aeruginosa. Sepa-rate analyses were carried out for the cell-associated and secreted proteins. LC-MS data were acquired using a nano-ACQUITY UPLC system and a QTof Premier mass spec-trometer operating in alternate scanning mode. In parallel, pull down experiments were carried out in which RNA spe-cies that co-purified with His-tagged RsmA were isolated, reverse transcribed, cloned and sequenced.



Comparison of the proteomics results and the pull down data revealed that majority of the proteins that were differentially expressed in the proteomics study were also identified among the pulled-down RNA species, including mRNAs encoding major virulence factors. These results suggest that RsmA regulates all of the differentially regu-lated genes directly by binding their cognate mRNAs. In addition, comparison of the proteomics and the pull down data showed that RsmA binds mRNAs of genes that are regulated either positively or negatively by RsmA, suggest-ing at least two different mechanisms for RsmA-mediated translational regulation.

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Mass Spectrometric Identification of Proteins of Purified Protein Derivative (PPD) of Mycobacterium Bovis Stain AN-5 Using a 2D HPLC Protein Fractionation SystemZ. Chen, Y. C. Chen, S. Krishnanchettiar, S. Gupta, S. S. Lateef, J. P. G.D.Lasanthi, B. Lee; University of Illinois at Chicago, Chicago, IL, United States.

Tuberculin PPD Bovis, Intradermic (purified protein derivative of Mycobacterium bovis strain AN-5) was widely used for the Tuberculin test in cattle. However, protein components of PPD remain unknown since it is extremely difficult to separate PPD proteins by traditional protein fractionation techniques. A pilot mass spectrometric pro-tein identification of PPD using the new protein fraction-ation system of Beckmen Coulter’s ProteomeLab PF 2D is reported here. ProteomeLab PF 2D is a two-dimensional liquid chromatographic system to fractionate complex proteomes. This system consists of a chromatofocusing column to separate the protein based on charge/pI in the first dimension and a reversed-phase column to separate the protein based on hydrophobicity in the second dimen-sion. The advantages of this technique include high load-ing capacity and better detection of hydrophobic and low molecular weight proteins. Up to 800 protein fractions are collected. So far, 50 proteins such as cfp10, MPB 53, MPB 70, and others have been identified using MALDI-TOF or FT-ICR MS with Sequest or Mascot search engines. This study has demonstrate that ProteomeLab PF 2D with mass spectrometry is a potentially powerful tool to identify pro-teins from a large proteome.

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DNA Damage Induces a Novel Interaction between Fanca and HuntingtinS. L. Conner, M. Wang; Indiana University School of Medicine, Indianapolis, IN, IN, United States.

The objective of this study was to elucidate the role that Fanconi anemia complementation group A protein (FANCA) plays in cells upon DNA damage through iden-tification of DNA damage dependent protein interactions with FANCA using the Tandem Affinity Purification (TAP) methodology. Fanconi anemia (FA) is a rare autoso-mal recessive disease that is characterized by aplastic ane-

mia, congenital abnormalities, birth defects and increased susceptibility to cancer. FA patients are assigned to one of twelve complementation groups, with about 65% of patients falling into complementation group A. FA patients share the common phenotypic feature of FA of bone mar-row failure, and cells from FA patient’s exhibit sensitivity to DNA crosslinking agents such as Mitomycin C (MMC) and Diepoxybutane. TAP was used in order to purify the protein complexes twice, and to maintain protein inter-actions based on post translational modifications. The TAP FANCA construct contains two tags, a streptavidin binding peptide (SBP) and a calmodulin binding peptide (CBP) which allow for the two-step purification. Human embryonic kidney cells (HEK 293) were transiently trans-fected with either a negative control or the TAP FANCA construct. Half of the FANCA transfected HEK 293 plates were treated with MMC twenty-four hours post transfec-tion to induce DNA damage. The transfected cells were col-lected forty-eight hours post transfection, and the FANCA interacting proteins were TAP purified and identified using Mass Spectrometry. MMC treatment induced a novel inter-action between FANCA and Huntingtin (HTT) that was not found in the untreated FANCA or negative control samples. The DNA damaged induced FANCA/HTT inter-action was confirmed through western blot. The role of the FANCA/HTT interaction in DNA damage is not known. More studies are underway to determine if this interaction is important for DNA damage recognition or repair.

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Simplified and Fully Automated Analysis of Complex Samples using LC-MSMSD. Cox, S. Tate; MDS Sciex, Concord, ON, Canada.

LCMSMS is now a standard method for the analysis of protein samples which have been separated by 1D or 2D gel electrophoresis with subsequent in-gel digestion. However, the throughput is low as the analysis requires the use of long gradients and run times of over 30 min per sample. A more appropriate method is to analyze the samples without gel fractionation but this workflow is hindered by the ability of modern mass spectrometers to efficiently collect MSMS on all available ions when coupled with modern LC sys-tems which produce very sharp chromatographic profiles. There is a growing trend towards resubmitting the samples after an initial analysis to collect data on ions which were initially missed.

Such a “repeat information dependant analysis” (rIDA) workflow is difficult to implement in an automated labora-tory as the resubmission of samples with modified method automatically is cumbersome. This poster highlights the usage of an automated method/batch submission system which allows for unattended rIDA analysis.

A dramatic increase in the number of proteins which can be identified in a given sample was accomplished by coupling this automated workflow with easy-to-use data-base searching software. This highlights that repeated anal-ysis of a sample consumed less material than using MudPIT based experimentation, and that a greater than threefold increase in the sample throughput can be achieved, without loss in quality of sample analysis using rIDA workflows.



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Keeping Proteins in Solution: Interval Free-Flow Zone Electrophoretic Separation of a Total Protein Extract from HeLa CellsK. Hartmann, M. Nissum, R. Wildgruber, G. Weber, C. Eckerskorn; BD Diagnostics, Martinsried, Germany.

The application of 2DE to membrane proteins is ham-pered by the limited protein solubility. In addition, the gel matrix is a limiting factor in combination with downstream analytical techniques such as mass spectrometry. Free-Flow Electrophoresis (FFE) in the isoelectric focusing (IEF) mode provides a carrier-free alternative to 2DE. IEF-FFE has proven useful for soluble proteins since the methodol-ogy is not limited in pH or size range. Various detergents have been applied to handle more hydrophobic proteins under denaturing conditions. However, a prerequisite for IEF-FFE is the maintained solubility of the proteins at their pI. This is not the case for many membrane proteins which have high tendency to precipitate at their respective pI.

Here we present Interval Zone FFE (IZ-FFE) as a novel mode of FFE which facilitates separation of charged molec-ular species including proteins and protein complexes. In contrast to IEF, the separation is carried out at a constant pH relying on the net charges of the proteins. The applied pH may be significantly different from the pI of the pro-teins to be separated thus maintaining proteins in solution that would otherwise precipitate. Resolution and solubil-ity may be increased further under denaturing conditions using urea and/or thiourea and by including non-ionic or zwitterionic detergents.

IZ-FFE was applied to a total protein extract from HeLa cells and compared to IEF-FFE. A significant increase in protein solubility was observed in the IZ-FFE mode. Sepa-rations were performed using detergent-free media at high loading rates without observing precipitation. This was in contrast to IEF-FFE where precipitation was observed even when detergents were included in the media. The detergent-free separation media facilitated the coupling to RPLC-MS/MS since no detergent removal was required and the proteins were maintained in solution.

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An in Vivo Extracellular Proteomic Study of the Human Fungal Pathogen Cryptococcus NeoformansR. A. Eigenheer, Jr., A. C. Gelli, B. S. Phinney; UC Davis, Davis, CA, United States.

The fungal pathogen Cryptococcus neoformans often causes life-threatening meningitis in immunocompromised individuals. Extracellular proteins are critical to the patho-genesis of cryptococcosis, and both secreted and cell wall-bound proteins contribute to this pathogenicity. Unfortu-nately, only a fraction of the C. neoformans extracellular proteome has been characterized to date.

To further elucidate the extracellular proteome of C. neoformans, we conducted an analysis involving both col-lection of secreted proteins and enzymatic (proteolytic) released proteins from the outer cell wall. Peptides were proteolyzed from live cells, then purified by offline high pressure liquid chromatography, and subsequently identi-

fied by LC-MS/MS. In order to assure only cell wall and secreted proteins were identified, Gray’s Reagent was used to stain cells and confirm membrane integrity. Cells were also treated with Brefeldin A, an inhibitor of protein traf-ficking through the classical ER/Golgi-dependent export pathway, and compared with untreated cells to assess secre-tion through this pathway.

Interestingly, a number of the proteins identified as cell surface in this study were present in the Brefeldin-treated cells; this extracellular presentation may imply non-classi-cal secretion. Also, a number of the proteins identified as extracellular in this study are generally considered cyto-solic; however, cytosolic proteins can be transported to the surface as a means of performing additional functions; these have been referred to as “moonlighting proteins.”

Unlike previous studies of either secreted proteins or proteins released from isolated cell walls, the novel use of proteases on whole cells in vivo without disrupting internal compartments allows for a more complete snapshot of the extracellular proteome of C. neoformans. Furthermore, this in vivo strategy may allow rectification of proteins misan-notated as intracellular. Finally, the Brefeldin A may give clues as to the route each protein takes to reach the surface of C. neoformans.

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Understanding Cisplatin Drug Resistance in Human Ovarian CancerD. P. Fitzpatrick, M. Wang; Indiana School of Medicine, Indianapolis, IN, United States.

Ovarian Cancer is the fifth leading cause of death from all cancers in women. Platinum-based chemotherapy is the primary treatment for ovarian cancer. Most patients with the disease are initially very responsive to chemotherapeutic treatment. However, the majority of ovarian cancer patients eventually relapses and becomes refractory to additional treatment. This drug-resistance is a major impediment to the successful treatment of ovarian cancer. To date the mechanisms of drug-resistance are poorly understood. To elucidate the underlying mechanisms in which drug-resis-tance is developed in ovarian cancer cells, global protein expression pattern changes in drug-sensitive and -resistant ovarian cancer cells need to be established.

In this study, we approached ovarian cancer resistance using proteomics technology to quantitatively profile the global protein expression levels of two pairs of ovarian cancer cell lines, with cisplatin drug treatment. A2780 and 2008 human ovarian cancer cell lines were chosen as cisplatin-sensitive and A2780/CP and 2008/C13*5.25 were their resistant counterpart. Statistical analysis was carried out by ANOVA models and these were fit using Proc_Mixed in SAS. Through our efforts to identify those protein changes associated with cisplatin drug-resistance, we identified and quantified over ~2000 proteins where 855 proteins were identified with high confidence (Priority 1) and 760 of them showed significant expression changes (False Discovery Rate is less than 0.05). Based on the data that we obtained, we were able to select a panel of potential proteins that could play a role in cisplatin drug resistance. Inhibition assays targeting select proteins from our panel



were carried out, to determine whether down-regulation of these proteins would be successful in reversing cisplatin resistance. Preliminary data showed a very promising effect in reversing the cisplatin resistance in the resistant cells when the combination of cisplatin and TETA, an inhibitor of antioxidant Superoxide dismutase (SOD1) protein, were used.

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Expression of c-kit, p-mTOR and p-NFKappaB in Angiolymphoid Hyperplasia with Eosinophilia: Potential for Therapy with Imitanib Mesylate, Rapamycin and BortezomibS. B. Forman1, R. E. Brown1,2; 1Geisinger Medical Center, Danville, PA, United States, 2Univeristy of Texas Medical School at Houston, Houston, TX, United States.

We report a case of a 54-year-old white female with a seven year history of angiolymphoid hyperplasia with eosinophilia. (Figures 1, 2) A proteomic evaluation of the tumor was performed with the intention of identifying targets for therapy. The tissue had high expressions of the following: platelet derived growth factor-alpha(PDGFR-α), platelet derived growth factor-beta (PDGFR-β), c-kit (CD 117), angiotensin converting enzyme, p-mTOR (SER 2448) and p-NF-κB p-p65.

The signal transducers PDGFR-α and PDGFR-β are expressed in the cytoplasm of numerous mast cells. c-kit, another signal transducer, is expressed in the plasmalem-mal compartment (cell membrane) by an appreciable num-ber of mast cells. The transactivator, ACE, is expressed in the plasmalemmal compartment of the lesional endothelial cells. The downstream effector, p-mTOR is expressed in the nuclear, cytoplasmic and plasmalemmal compartments of the lesional endothelial cells. And, an antiapoptotic/tumor-igenic factor, NF-κB p-p65 is expressed in the nuclear and cytoplasmic compartments of endothelial growth cells.

Imitanib mesylate (Gleevec) targets c-kit, PDGFR-α and PDGFR-β. ACE inhibitors, such as enalapril, target angiotensin converting enzyme. Sirolimis (Rapamycin) antagonizes p-mTOR whichinhibits mast cells and lym-

phocytes via reduced IL-5 and vascular endothelial growth factor (VEGF). Bortezomib (Velcade) blocks the activation of NF-κB which interrupts VEGF production and endo-thelial cell stimulation.

P159-T

Computer Analysis of 2D GelsM. F. Goldfarb; Anatek-EP, Portland, ME, United States.

This work identifies statistical algorithms which need to be included in analysis of 2D gels for accurate determi-nation of differential spot intensity changes. Two-dimen-sional electrophoresis is a powerful tool for determining differential protein expression in complex mixtures, but 2D electrophoresis, to date, is not producing sufficiently reliable results due to the degree of gel variability generated by this methodology. The new DIGE procedure, compar-ing two samples in the same gel, does eliminate some of the variability introduced with gel to gel comparison, but still has variability due to difference in dye binding, charge, and fluorescence. Introducing quality assurance statistical algorithms is necessary to extract meaningful data from the gels. A quality control analysis of replicate gels needs to be performed prior to using the set in the final analysis. Increasing replicates to five from the usual three can only add greater variability. A statistical “replicate quality” gel test needs to be done on the computer gel images, and repli-cates with greater than 20%–30% variability should not be used. In addition, since spot intensity data is not normally distributed, spot differential determinations cannot be a t test. The Studentized Range has been suggested as a more accurate method for calculating significant difference.

P160-S

Identification of Glutamine-Induced O-GlcNAc Modified Proteins in a Model of Critical IllnessK. R. Jonscher, S. Pinto, C. Hamiel, P. Wischmeyer; University of Colorado HSC, Denver, CO, United States.

Enhanced heat shock protein (HSP) expression pro-tects cells and tissues from injury and HSP expression can figure 1

figure 2



improve survival in experimental illness models. Our labo-ratory has shown that treatment with glutamine (GLN) can enhance HSP expression in tissues of stressed animals. An important part of GLN’s protective effect may be via rapid O-glycosylation of key cellular proteins, potentially via enhanced activity of the hexosamine pathway follow-ing stress. This is known to occur in seconds, prior to the expression of stress proteins, and can prevent proteosomal degradation of these vital proteins. The activation of the O-GlcNAc pathway is known to induce a cellular protective response, including enhanced HSP expression which may serve as a nutritional sensor of the cell’s environment.

In this study, we pre-treated HSF1 wild-type cells with DMEM + 10% FBS for 21 h and 20 min. Cells were treated with either 50 mM glutamine or 5 mM glucosamine 15 min prior to heat shock. After 4 h, cells were fractionated into nuclear and cytoplasmic fractions and lysed. Anti-O-GlcNAc was used to immunoprecipitate modified proteins from the lysates, which were then separated using 1D gel electrophoresis. Bands showing changes were excised, digested and identified by nanoLC/MS/MS and database searching. The majority of protein changes were observed in the nuclear fraction, where treatment with glutamine, and to a lesser extent glucosamine, rescued some heat shock proteins to near control levels of expression. As expected, in our preliminary work, the expression of O-GlcNAc modi-fied HSP-70 was at similar levels for control and glutamine-treated heat shocked cells, while expression was reduced for untreated heat shocked cells. Other modified proteins detected include myosin, plectin 1, alpha filamin, vimen-tin and actin. Future efforts will include replicating these experiments in cell and mouse models to confirm these results.

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De Novo Peptide Sequence Database for Protein IdentificationM. Kanazawa1, S. Egawa1, H. Anyoji1, Y. Hoshino2,3, U. Nagashima4; 1Reifycs Inc., Tokyo, Japan, 2Leave a nest Co., Ltd., Tokyo, Japan, 3University of California, Irvine, CA, United States, 4National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan.

Several protein identification methods using mass spectrum are utilized for proteomic analysis, and almost all methods rely on protein sequence databases for the characteristic of their algorithms. Basic algorithm of these methods is to compare molecular weight (MW) of peptide as digested protein or dissociated peptide fragment with calculated weight by database stored sequences and to iden-tify which peptide or protein is measured.

The number of human protein entries in Uniprot data-base (UniprotKB / Swiss-Prot Release 9.0), for instance, is 14,987, and it does not reach even the number of human gene. In short, identified protein by database oriented pro-tein identification methods are limited because of the vari-ety of known protein sequences, a lot of mass spectrum is hard to lead identical peptide sequence.

We, therefore, propose the application of de novo peptide sequence databases for protein identification. De novo peptide

sequences here are constructed by computational calculation not experimental process. As peptide sequence is basically constructed by the combination of 20 kinds of amino acids, if sequence length is n, 20n kinds of sequence are estimated. It is naturally that the data size of estimated sequences and their MWs are too huge to store in practical disk capacity, but we developed data storing scheme particular for peptide sequence fragment using table driven approach.

De novo sequencing approach is localized sequence identification because it reads sequence by not sequence database but only the intervals of peak combination. It is suitable for the point at this discussion, but hard to iden-tify the sequence without clear distinctions of measurement peaks. On the other hand, this presenting database is for global sequence identification that almost all protein iden-tification method applies. In this presentation, we show the contents of database and its application with several examples.

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Plasma Proteomics of Colon Cancer Patients—The Individual Regulation of Protein Isoforms Identified by the ICPL-TechnologyE. Keidel1, A. Brunner1, T. Halder2, D. Suckau3, S. Martin4, J. Kellermann1, F. Lottspeich1; 1Max Planck Institute of Biochemistry, Martinsried, Germany, 2Toplab GmbH, Martinsried, Germany, 3Bruker Daltonik GmbH, Bremen, Germany, 4Blood Donor Centre Bavarian Red Cross, Munich, Germany.

Already before the occurrence of pathological symp-toms in the development of severe diseases changes in the proteome pattern in the plasma of patients can be observed. Therefore, to follow these pathological changes, plasma samples from several time points should be analysed before clinical symptoms arise. Future patients can be statistically expected within the pool of periodical blood donors. From the biobank (blood donor centre of the Bavarian red cross) colon cancer patients can be derived out of a pool of nearly 300,000 donors every year.

Within this project (20 individuals) plasma samples of five different time points, collected each from the same individual were depleted from 12 high abundant proteins, labelled with ICPL and separated by 2D-gelelektrophoresis. Proteins were stained with Sypro-Ruby, picked and enzy-matically cleaved with Trypsin. Peptides and corresponding proteins were identified by MALDI-MS and MS/MS anal-ysis. From several gene products up to 30 different isoforms could be found, many of them differentially regulated.

These isoforms, usually derived from one single gene, are produced due to splicing, degradation and posttransla-tional events. Importantly, they would not have been dis-criminated if GIST or if shotgun proteomics approaches would have been used. Applying these methods, the pro-teome would have been cleaved enzymatically before label-ling. After enzymatic cleavage of similar protein species their peptides could not be reliably used for quantification since they may be obtained from differently regulated pro-teins. Only a few proteotypic peptides (which are specific for a single protein species) are suited for accurate quanti-



fication. However, with usual sequence coverage of far less than 50% chances are quite high to miss these peptides.

During the ICPL approach the stable isotopic label is already introduced on protein level, thereby bypassing the disadvantages of GIST approaches and maintaining the benefits of correlating protein modifications with their regulation.

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A Density Based Sample Fractionation Technology:Folate Deficiency (FD) and Oxidative Stress Response in Brain and LiverW. Lan1, J. Guhaniyogi1, M. J. Horn1, B. Graham2, J. Q. Xia2; 1Prospect Biosystems, LLC, Newark, NJ, United States, 2Pel-Freez Biologicals, Inc., Rogers, AR, United States.

Folate deficiency (FD) alters hepatic methionine metabolism and is associated with increased hepatocellu-lar apoptosis. Additionally, mice deprived of folate showed increased oxidative damage in brain tissue leading to cogni-tive impairment. Most previous studies have focused inde-pendently on either liver, the main tissue of folate storage and metabolism, or brain, where folate regulates neurogen-esis and programs cell death. The aim of this study was to apply a powerful and rapid proteomics approach to under-stand potential subcellular correlations of folate deficiency in both brain and liver of the same rat. This approach com-bined a new density based sample fractionation technology (Edge Technology) with other conventional proteomics techniques, such as western blot analysis, 2DE and mass spectrometry. The brain and the liver from individual rats, fed normal or FD diets for 6 weeks, were homogenized and then fractionated using the Edge 200 Separation System. Subsequently, all fractions from brain and liver, from con-trol and treated rats, were analyzed by western blot using two markers of oxidative stress: glutathione peroxidase 1 (GPx1) and Glucose-Regulated Protein 75 (GRP75). Cer-tain fractions were selected based on western blot analysis and were further analyzed by 2DE. Protein spots of interest were identified by MALDI-TOF/TOF. The results demon-strated that Edge technology provides a powerful density based separation and enrichment method for rapid screen-ing of potential FD markers and their possible correlations to both liver and brain diseases.

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Optimization of the Gel Shredder: A Centrifugal Device for Automated In-gel Digestion with Improved Peptide Recovery.A. Lazarev, T. Rejtar, B. L. Karger; Barnett Institute, Northeastern University, Boston, MA, United States.

Proteomic workflow frequently involves gel separations. Poor peptide recovery from in-gel digestion procedures is at large influenced by the properties of polyacrylamide gel, which restricts access of proteolytic enzymes to the protein substrate as well as prevents complete extraction of certain peptides. We have designed an optimized centrifugal device

which allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, roughly 100 µm, prior to the enzymatic digestion. Such treatment increases the surface area of gel pieces and allows the enzymes and solvent to better penetrate the gel lattice. The performance of such novel device has been evaluated using several gel-separated model proteins pre-labeled with thiol-specific multiplexed stable isotope tags, followed by relative quantification by a MALDI-TOF/TOF mass spectrometer. The performance of the new device, called the Gel Shredder, has been compared to standard in-gel digestion protocols. It has been shown that an improvement in peptide recovery can be reproducibly obtained by using the Gel Shredder devices. It has been determined that the application of the Gel Shredder is particularly useful for recoveries of high molecular weight hydrophobic peptides, presumably due to their higher affinity to the polyacrylamide gel lattice. Gel Shredder also allows to save time usually spent on gel band manipulation during in-gel digestion experiments.

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Mass Spectrometric Identification of Proteins of the Ureaplasma Urealyticum Using a 2D Chromatographic Protein Fractionation SystemB. Lee, S. Gupta, S. Krishnanchettiar, Y. C. Chen, S. S. Lateef, J. P. G.D.Lasanthi, Z. Chen; University of Illinois at Chicago, Chicago, IL, United States.

A pilot protein profiling of one of the smallest organ-ism ureaplasma urealyticum, a pathogen causing urogeni-tal and placental infection/inflammation, using the new protein fractionation system of Beckmen Coulter’s Pro-teomeLab PF 2D is reported here. ProteomeLab PF 2D is a two-dimensional liquid chromatographic system to fractionate complex proteomes. This system consists of a chromatofocusing column (250 × 2.1 mm) to separate the protein based on charge/pI and a nonporous reversed-phase column (4.6 × 33 mm) to separate the protein based on hydrophobicity. The advantages of this technique include high loading capacity and better detection of hydrophobic and extreme PI and molecular weight proteins. Up to 1000 protein fractions are collected. So far, 30 proteins such as GroEL, heat shock protein, protein L7/L12, and oth-ers have been identified using MALDI-TOF or FT-ICR MS with Sequest or Mascot search engines. This study has demonstrate that ProteomeLab PF 2D with mass spectrom-etry and some precautions is a potentially powerful tool to identify proteins from a large proteome.

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Novel Method for High-Throughput Cross-Linking Sites AnalysisY. Lee, B. S. Phinney, J. M. Nunari, L. L. Lackner; University of California, Davis, CA, United States.

We developed a new strategy and computer algorithm for high-throughput analysis of protein-protein or intra-protein interaction sites from chemically cross-linked protein(s). In this strategy, we directly analyze LC-MS/MS



data obtained with LTQ-FTMS without any comparison with control sample or isotopic labeling. We developed a computer algorithm, X!Link, to find cross-links of two peptides, which takes only ~10 seconds to analyze ~5000 MS/MS spectra. It is very sensitive and has low false-positive rate. We applied this method for the cross-linking site analysis of cytochrome c and Dnm1 G385D homodi-mer chemically cross-linked by BS3. We also adopted X!Tandem and Sequest for intra-peptide cross-linking and monolink modification analysis. Total of twenty one cross-links are identified in a single LC-MS/MS data of cyto-chrome c, in which thirteen is inter-peptide cross-links in 42 MS/MS spectra. High coverage of BS3 modified Lys (84%, 16/19) is owing to the high sensitivity of the present method. Monomer and dimer SDS-PAGE bands of Dnm1 G385D were studied to investigate inter-protein interac-tion sites of this homodimer. Total of forty five cross-links including thirty seven inter-peptide cross-links from 243 MS/MS spectra were identified in four LC-MS/MS datas-ets, which is the most cross-links identified so far in a single protein. Closer look of the data demonstrates importance of careful manual inspection for the correct assignment of cross-linking sites when multiple sites exist in a peptide or other similar sequences exist. Two such cases are shown in Figure 1.

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Microwave-Assisted Acid Proteolysis of ProteinsP. S. Liu, W. Sandoval, J. R. Lill; Genentech, Inc., South San Francisco, CA, United States.

Introduction: Microwave-assisted acid hydrolysis has recently been reported in the literature as a tool for bot-tom-up proteomic analysis as well as increase coverage of proteins when traditional proteolytic cleavage sites are not present, and also for identification and characterization of the termini of proteins. Here two microwave-assisted acid proteolysis methods were compared, one involved the use of trifluoroacetic acid (TFA) and the other with formic acid. In this experiment, we monitored the; acid cleavage speci-ficity; reaction / preparation time overall protein coverage; and N- and C-terminal coverage.

Materials and Methods: Standard proteins (1–5 µg) were diluted into two acids 1–5 M TFA and 1%–50% formic acid, which were subjected to microwave irradiation using an industrial microwave system (CEM Corp., Matthew, NC). Samples were microwave irradiated for 1–5 min and resulting peptide fragments were then subjected to reverse-phase separation, followed by MSn analysis on an LCQ-Deca Ion Trap mass spectrometer (Thermo Electron, San Jose, CA) fragmenting the three most intense peaks. MS/MS data were searched with Mascot (Matrix, London) for protein coverage and identification with ‘no enzyme’ selected in the search parameters.

Results: Preliminary results show that controllable cleavage at specific proteolytic sites can be obtained when employing set acid concentrations under controlled micro-wave irradiation. As previously reported, formic acid can cleave preferentially at Aspartic acid. TFA can be manipu-lated to cleave at either preferentially the N and occasion-ally C-terminus of a protein, with longer incubation times leading to internal cleavage at acid labile bonds.

Conclusion: Microwave-assisted acid proteolysis can be used either as an alternative proteolytic reagent with less sequence constraints than traditional enzymes. Conditions can be manipulated to direct proteolysis to mainly the ter-mini of the protein, or indeed internal cleavage sites.

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High Resolution Gel-based Analysis of Differentially Expressed Membrane Proteins of CD4+ T Helper CellsP. Lutter, A. Wattenberg, V. Veneruso, M. Blueggel, S. Muellner; Protagen AG, Dortmund, Germany.

CD4+ T helper cells are the key player for control and regulation of adaptive immune responses. In a healthy immune system they protect the body against a variety of pathogens but CD4+ T cells are also involved in pathogen-esis and perpetuation of diverse autoimmune diseases like chronic rheumatic inflammation. Consequently, analysis of T cell activation has the potential to assist the development T cell-directed therapies and diagnostic approaches. Mem-brane proteins are of specific interest because they represent 60% of known drug targets and have also the potential to serve as diagnostic markers.

figure 1

Top:Twointernallysresiduesinapeptide.Bottom:exactlythesameparentmassbetweencross-linkedpeptidesoftwosubsequentpeptidesequencesandasinglepeptidewithmonolinkmodification.



The membrane proteomes of a well characterized set of freshly isolated and CD3/CD28-stimulated human CD4+ T cells were analyzed by high resolution 2D-IEF-SDS-PAGE. Membrane proteins were enriched by differential centrifu-gation and following Triton-X114 extraction. Reproducibil-ity of this isolation method for membrane proteins and 2D PAGE was proven by comparison of proteomes of resting and activated CD4+ T cell samples derived from individual human donors. Using differential centrifugation and deter-gent extraction protein spots were displayed in proteomes that were not detectable or underrepresented in 2D gels of whole cellular lysates without any fractionation.

Furthermore, for quality control of the procedure a selection of well known surface antigens was quantitatively measured by FACS analysis prior to electrophoresis. These antigens were stained by large 2D-PAGE western blot of the separated T cell proteins identified by mass spectrom-etry. They were assigned to the corresponding 2D gel maps for quantitative spot analysis. Recovery as well as expres-sion ratios of surface antigens between resting and acti-vated T cells were compared.

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Rapid and Sensitive Identification of Amyloid Proteins from Formalin-Fixed Paraffin-Embedded Sections and Fat Aspirates: Potential for Aiding in Clinical Diagnosis of Systemic AmyoidosisB. J. Madden, J. A. Vrana, J. D. Gamez, A. Dogan, H. R. Bergen; Mayo Clinic College of Medicine, Rochester, MN, United States.

Human amyloidosis describes a condition where one of some 23 known proteins form non-soluble pathological fibrils in various organs and tissues throughout the body. A common feature of these amyloid deposits is distinctive green birefringence when stained with Congo Red. Stain-ing is performed on formalin fixed paraffin embedded (FFPE) tissue sections of tissue biopsies or on subcutane-ous abdominal fat aspirates. This method is combined with clinical, immunohistochemical, and genetic information to diagnose the condition and in some cases, identify the amyloid involved. Definitive identification of the amyloid protein is essential for proper therapies in treating patients. Identification in the cases where the other methods give ambiguous results, involve solubilizing and extracting the amyloid proteins from the fixed sections or from fat aspi-rates. In an attempt to reduce the processing time of these types of clinical samples and still provide definitive identi-fication, we explored in-situ enzymatic digestion methods that exploit the speed and sensitivity of nanoRPLC-ESI-tandem mass spectrometry. Using previously diagnosed clinical samples, we tested a variety of conditions to perform trypsin digestion directly on laser capture microdisected (LCM) generated samples and on fat aspirate samples with the goal of having a robust method suitable for mass spec-trometry based identification. Trypsin digestions were per-formed using, ammonioum bicarbonate, Tris, Tris EDTA/ ± zwittergent 3-16, with and without solubilization steps of hexafluoroisopropanol, acetonitrile, or DMSO. A commer-cially available FFPE solubilization kit, Liquid Tissue, was also tested. The efficacies of these treatments are judged by

performing Sequest or Mascot searches of LTQ generated MS/MS spectra, noting the presence of peptides from the expected amyloid proteins. This method has been success-fully utilized to correctly identify amyloids comprised of transthyretin (TTR), serum amyloid A (SAA), lambda and kappa light chains. Controls taken from regions adjacent to the amyloid plaques were consistently devoid of the amy-loid proteins.

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Platform for Proteome Analyses of LC/MS/MS Data by Multiple Search Engine Comparison and Label-Free Relative QuantificationM. A. McFarland, D. J. Slotta, S. P. Markey; NIMH/, Bethesda, MD, United States.

LC-MS/MS and automated protein library searching provide a high-throughput strategy for peptide sequence assignments for identification of qualitative differences in comparative shotgun proteomics. However, the rapid increase in size and depth of analysis requires advancement in the speed, scale and flexibility of tools available for par-simony level data analysis. MassSieve has been developed as a platform for parsimony analysis of large scale MS/MS experiments, both for single and comparative analysis.

MassSieve supports reports from multiple search engines with differing probability-based search character-istics which can increase peptide sequence coverage and/ or identify conflicting or ambiguous spectral assignments. Label-free relative quantitative information is also available by spectral hit counts per peptide and per protein. Graphi-cal display of each set of related peptides and proteins as well as user defined treatment of indeterminate peptides provides for visualization of possible isoforms or conflicting database entries.

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Examination of Non-Specifically Bound Proteins and Peptides Following Depletion of Twenty High-Abundance Proteins from Human PlasmaC. Melm, M. Schuchard, A. Crawford, H. Chapman, K. Ray, D. Chen; Sigma-Aldrich, St. Louis, MO, United States.

Depletion of high abundance proteins from human plasma and serum has become a valuable tool in the search for protein biomarkers. However, some speculate the exis-tence of potential biomarkers associated with the depleted proteins. For example, human plasma albumin is a well-known carrier of proteins and peptides. Previous studies have identified a limited set of non-specifically bound proteins associated with proteins removed from serum and plasma, but the dynamic range of the depleted fraction may affect its accuracy and content. Therefore, an exhaustive analysis of the depleted proteins is important to address this challenge. Different elution methods and gradients will be evaluated to selectively elute non-specifically bound proteins and peptides from the depletion resin without co-eluting the twenty, targeted proteins. The identifications



from this analysis should produce a more complete set of non-specifically bound proteins and peptides.

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Intelligent MS/MS Acquisition Results in Higher Sequence Coverage and More Confident Protein IDs from Complex SamplesJ. E. Meza1, J. Dojahn2, V. Kruft2; 1Applied Biosystems, Foster City, CA, United States, 2Applied Biosystems, Darmstadt, Germany.

A major challenge in the study of proteomics is the anal-ysis of complex samples. As sample complexity increases so does the need for a highly sensitive instrument with large dynamic range capabilities. Also required for deeper analysis of complex samples is the ability to perform high speed MS/MS scans without loss of spectral quality during an LC-MS/MS run. In the current study using a QqTOF instrument, we used a new feature that enables dynamic acquisition time of MS/MS spectra based on the evaluation of fragment ion intensity in combination with repetitive LC-MS/MS runs that exclude previously acquired precur-sors. Utilizing this intelligent MS/MS acquisition feature we achieved low attomole level sensitivity and four-log orders of dynamic range on a commercially available sam-ple. Analysis of more complex samples (i.e. animal tissue) resulted in the identification of hundreds of nonredundant proteins from single cation exchange fractions with greater than 95% confidence using 45 minute gradients. The use of dynamic acquisition time and the repetitive LC strategy combined with ProteinPilot software illustrate the effec-tiveness of our approach in identifying a large number of nonredundant proteins with high confidence in a relatively short time.

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Slippery when Translated: Extensive Programmed Ribosomal Frameshifting Revealed by ProteomicsR. Ogorzalek Loo1, Y. Yang1, Y. Xie1, J. Dunsmore1, H. Mouttaki2, M. McInerney2, R. Gunsalus1, J. Loo1; 1University of California, Los Angeles, CA, United States, 2University of Oklahoma, Norman, OK, United States.

Living organisms translate genetic information in many ways, occasionally altering transcription signals and chang-ing reading frames. A consequence of these post-transcrip-tional actions (recoding) is that a single coding sequence can produce multiple gene products, or a single product from two overlapping open reading frames (ORFs). Although examples of recoding have been uncovered in all well-stud-ied organisms from viruses to bacteria to humans, we know little about the global contribution recoding makes to pro-teomic complexity. Proteomic methods tailored to expose recoding events will have enormous impact.

In examining the proteome of Syntrophus aciditrophicus, a gram-negative bacterium that grows syntrophically (coop-eratively) with methanogens or sulfate reducers, we found several examples of translational frameshifting, apparently arising from particularly “slippery” mRNA sequences (X

XXY YYZ). De novo sequencing of LC-MS/MS spectra obtained from trypsin-digested 2D gel spots reveals multi-ple peptide sequences arising from a single stretch of mRNA sequence, providing a rare opportunity to view proteins generated in vivo by recoding. Linking sequenced peptides to 2D gel spots presents the extraordinary opportunity to monitor ratios of recoding products with respect to changes in culture conditions and even whether S. aciditrophicus is cultivated syntrophically or in isolation.

Our studies revealed that predicted 4- and 16-kDa ORFs actually correspond to a 30-kDa product with at least two frameshifts (peptides identified in all 3 reading frames). More than ten tryptic peptides are observed that do not correspond to the primary sequence in any single reading frame. Instead, they define the actual recoding site.

These data extend the questions about small ORFs beyond “real or unreal?” to “small or enlarged with frame-shift?” They also promise to elucidate factors regulating frameshifts, translational bypassing, and readthrough. Finally, they raise important questions about present capa-bilities to predict and quantify gene products, and future capabilities needed to address these issues in high-through-put.

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Affinity Protein Purification by Automation Using a Magtration Robotic SystemS. Patel1, X. Duan1, D. TerBush1, Q. Yang1, K. Obata2; 1PSS Bio Instruments Inc., Gaithersburg, MD, United States, 2PSS Bio Instruments Inc., Livermore, CA, United States.

Affinity purification is a powerful tool for protein enrichment in proteomics studies. We here present a fully automated system for purification of His-tag proteins and IgG using Ni2+/Co2+ and Protein A magnetic beads, respec-tively. Reagents for His-tag protein or IgG purification are pre-dispensed in a sealed cartridge for automated runs on a Magtration 12GC robot. The automated purification is based on Magtration technology to perform magnetic bead separation similar to a filtration process in a pipette tip. An optimized protocol has been developed for the automated protein purification. High protein purity and yields were obtained using this automated system. His-tag protein human galectin-1 was purified to approx. 1.6 mg with 12 samples processed in parallel within 30 min on the 12GC robot. This system was also used to screen the expression of His-tag water-soluble proteins and inclusion bodies in bacterial cells, even at a very low expression level. Using Protein A magnetic beads and corresponding pre-filled reagent cartridges, various amounts of human serum (15-80 µL) and the magnetic beads (100–200 mg) were tested on the robotic system. With 30 µL serum and 150 mg mag-netic beads, we purified IgG with a high yield of 230 µg. A total of approx. 2.8 mg IgG can be obtained within 60 min with 12 samples run in parallel on the robot. The magnetic beads after the affinity purification can be regenerated by automation for repeated use. Magtration robotic system can be extended for purification of GST-tag fusion pro-teins and Immunoprecipitation by automation. We have provided an automated protein purification system with a



Magtration robot and pre-filled reagent cartridges for rapid and multiparallel processing of different proteins.

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Enrichment and Detection of Molecules Secreted by Tumor Cells Using Magnetic Reversed Phase Particles and LC-MALDI-TOF-MSJ. F. Peter1, A. M. Otto2, B. Wolf2; 1Steinbeis Center for Cell Chip Technologies, Munich, Germany, 2Technical University of Munich, Munich, Germany.

Tumor cells are changing their genetic expression pattern as they progress to states of increasing malignancy. Investi-gations at the DNA and RNA level alone do not provide all the information resulting after the translation and process-ing of the corresponding proteins, which explains reports on a poor correlation between mRNA and the respective protein abundance. Unfortunately, peptides produced by cancer cells are secreted only in very low amounts, mak-ing mass spectrometric determination very difficult. In this work, methods have been evaluated for the effective enrich-ment and cleanup of substances secreted by cancer cells. To avoid peptides from fetal calf serum, a serum surrogate was developed, which do not alter the growth rate of the cancer cells. After binding of substances from cell culture supernatants with own manufactured magnetic reversed phase particles, the substances were eluted and separated by capillary HPLC. Fractions were spotted directly on a MALDI-target and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode.

During the investigation of two mamma carcinoma cell lines (MCF-7, non invasive, and MDA-MB231, invasive), unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensi-tive investigation of peptides secreted by cancer cell lines and will be used in the future for the investigation of cancer cell lines in different states of malignancy.

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Rapid Purification and Enrichment of Proteins from Crude Solutions Using Reversed Phase Microparticles and Subsequent Generation of Peptide Mass Fingerprints by MALDI-TOF Mass SpectrometryJ. F. Peter1, B. Wolf2; 1Steinbeis Center for Cell Chip Technologies, Munich, Germany, 2Technical University of Munich, Munich, Germany.

The generation of peptide mass fingerprints followed by a database search is a common tool for the mass spectro-metric identification of proteins. To provide a high sensitiv-ity, proteins must be efficiently purified and concentrated prior to enzymatic digestion. Common desalting proce-dures, like ultrafiltration or dialysis, are very time consum-ing and work best for high protein concentrations. Another critical point is the proteolysis of the investigated protein, which works efficiently only with concentrated protein solu-tions. Therefore, efficient concentration and simultaneous purification using solid phase extraction (SPE) will be the

method of choice to receive pure and highly concentrated protein solutions prior to enzymatic digestion. In this work, we manufactured magnetic reversed phase particles for the efficient purification and simultaneous concentration of protein samples with volumes up to several millilitres. The SPE procedure was compared with dialysis using commer-cial available microconcentrators with a cut-off membrane. Due to the magnetic core, each washing and elution step could be performed within 15 minutes. Then, the bound protein was digested directly on the beads, resulting in a remarkable increase of protein detection and better mass structural analysis. Useful MOWSE scores were achieved using bovine serum albumin as a model protein with con-centrations as low as 50 ng / ml (720 pM). Compared to the dialysis procedure, which needs several hours, the isola-tion and purification of protein can be performed in min-utes with the reversed phase particles.

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In Vivo Proteomics in Drosophila melanogaster by Tandem Affinity Purification of Protein ComplexesJ. S. Rees1, N. Lowe2, J. Howard1, S. Hester1, D. St. Johnston3, K. Lilley1; 1Cambridge Center for Proteomics, University of Cambridge, Cambridge, United Kingdom, 2Gurdon Institute, University of Cambridge, Cambridge, United Kingdom, 3Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.

In order to characterize in vivo interactions in Dro-sophila melanogaster, and further our understanding of developmental processes, hybrid PiggyBac/P-element YFP traps with Strep Tag affinity tags were generated to isolate multi-protein complexes. The incorporated Strep affinity tag allows the target protein to be isolated from its native environment, by tandem affinity purification, along with any associating proteins. Purification efficiency can be fol-lowed visually by the YFP marker. Complex proteins are then identified by tandem mass-spectrometry against the Drosophila melanogaster database using the Mascot search engine.

Due to the vast number of putative proteins, a high throughput purification and identification system has to be adopted to maximize coverage. Protein extraction tech-niques have been optimized to include isolation of tagged membrane proteins whilst maintaining their native struc-ture and complexes. Strep-Tactin columns have proven to be inefficient at eluting high enough yields of Strep-tagged purified protein thus making the purification time con-suming and tedious. However, modifying elution protocols has enabled us to load eluates directly onto the Mass Spec and thus identify all proteins in a single run.

The addition of a FLAG tag will also greatly improve the purification efficiency. Vectors have been designed and new fly lines are underway.

Mass Spec results were confirmed by visualizing the corresponding sized proteins on SDS-PAGE gels and west-ern blotting using a GFP antibody to identify the tagged bait. Data from this analysis will be uploaded onto a world-wide accessible database and supersede data generated from the yeast two-hybrid interaction screens.



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Profiling the Phosphoproteome: Discovery of Treatment Dependent Protein Markers of p38 and MK2 InhibitionT. J. Strelitzer1, S. E. Warder1, D. C. Perron1, S. S. Kwak2, J. S. Kamens2, M. J. Tomlinson2, J. C. Rogers1; 1Abbott, Abbott Park, IL, United States, 2Abbott, Worcester, MA, United States.

Protein phosphorylation is a critical signal transduction event in many areas of therapeutic interest, including can-cer and inflammation. Understanding of these signaling events, their role in disease, or their modulation by therapy, necessitates identification and characterization of phos-phoproteins regulated by hormones, circulating factors, or pharmacological agents. Identification of regulated phos-phoproteins requires sensitive and robust methods of phos-phoprotein enrichment, profiling, identification, and char-acterization. In this study we utilized a well-characterized cellular inflammation model, LPS-stimulated and drug treated THP-1 cells, to quantitatively assess the enrichment of known and total phosphoproteins with a commercial phosphoprotein enrichment kit. We measured the enrich-ment of Hsp27 with pan- and phospho-specific antibodies using flourescence based assays and 1D/2D Western blots, and measured total phosphoprotein enrichment with two phosphoprotein quantitation kits. These enriched fractions were then processed and profiled by 2D-DIGE to iden-tify known and unique regulated phosphoproteins. Several phosphoprotein candidates were characterized by 1D- and 2D-Western blotting to verify regulation. Our results indi-cate that current phosphoprotein enrichment reagents are: 1) subject to poor recovery and preferential enrichment of multiply phosphorylated proteins; 2) that sample processing (desalting) of enriched intact proteins for 2D gel analysis can lead to significant sample loss and artifactual regula-tion, and 3) that verification of regulation by 1D and 2D Western blotting is an essential step in the identification of phosphoprotein markers of interest.

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A Verification of Novel Experimental Design for Comparative Two-Dimensional Gel Analysis: Pre-Labeled Samples Fractionated on Anion Exchange Column Followed by 2D DIGEV. C. Ruddat1, P. Beckett2, C. R. Rozanas2, G. R. Asbury2; 1GE Healthcare, Sunnyvale, CA, United States, 2GE Healthcare, Piscataway, NJ, United States.

A common issue in Proteomics is that analytical meth-ods of separation do not have sufficient resolving power to deal with the tremendous sample complexity normally associated with a proteomics experiment. While 2D elec-trophoresis is superior to other methods in this respect, pro-teins can still co-migrate. Different methods of pre-frac-tionation have been employed in the past to rectify this problem. Recently, we demonstrated that E. coli samples pre-labeled with Cy2, Cy3, and Cy5 and fractionated on an anion exchange column resulted in reproducible fractions, without introducing any bias associated with the pre-label-ing. To further verify the reliability of this pre-fractionation

method for quantitative purposes, we spiked four different amounts of 5 proteins (not native to E. coli) into the sample. The fractions were run on an anion exchange column and subsequently resolved with 2D electrophoresis. The DIGE gels were analyzed with DeCyder 2D v. 6.5. The results from this study show a very close correlation between the expected theoretical results and the actual experimental results.

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In-Depth Evaluation of Data Acquisition Modes on High-Performance FT-MS InstrumentationM. Scigelova, G. Woffendin; Thermo Fisher Scientific, Hemel Hempstead, United Kingdom.

The hybrid FT mass spectrometers (i.e., LTQ Orbitrap) can be used in several different data acquisition modes. The accurate mass measurement of the precursor peptide ion is a prerequisite for confident protein identification, and should not be compromised. But MS/MS fragmentation spectra can be acquired using either an ion trap (low resolution/low mass accuracy—fast) or an orbitrap (high resolution/mass accuracy—slower) analyser as a detector. Moreover, dif-ferent fragmentation techniques can be employed, such as collisionally induced dissociation (CID) (in an ion trap) or higher energy collisional dissociation (HCD) (in a C-trap). One would expect these parameters to impact significantly on the results of protein identification by database search-ing or de novo sequencing.

We carried out an extensive evaluation of the datasets acquired on LTQ Orbitrap using various method settings. The particular questions we addressed in this study were:

Which method delivers maximum sequence coverage for detected proteins?

Which method provides data most suitable for auto-mated de novo sequencing?

What is the performance of PEAKS for automated de novo sequencing?

These questions were answered in the context of LC-MS analyses of a protein digest sample of medium com-plexity (six proteins).

The results showed that performing MS/MS fragmen-tation in the ion trap (CID with the ion-trap detection) and in the C-trap (HCD with the orbtitrap detection) are roughly equivalent with respect to the total protein sequence coverage obtained. However, when automated de novo sequencing is to be employed, the latter method afforded far superior results. Twice as many complete cor-rect sequences were obtained for HCD data compared to LTQ fragmentation spectra.



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A New Software Tool to Facilitate False-Positive Rate Assessment in Protein IdentificationS. L. Seymour1, E. Schreil2, W. H. Tang1, A. Loboda1, D. A. Schaeffer1; 1Applied Biosystems, Foster City, CA, United States, 2Applied Biosystems, Überlingen, Germany.

There are currently many employed variants of a class of methods for assessing the false-positive rates in protein identification results, referred to generically here as “decoy database searching.” The general idea of all variants is to assess false-positive rates by presenting identification soft-ware with some component of potential answers that are known to be incorrect. The major variants in this class of methods differ primarily in (1) the type of decoy compo-nent (reversed proteins, randomized sequences, or other more sophisticated approaches) and (2) whether the valid sequences and decoy component are searched separately or together competitively. Regardless of which specific approach is preferred, it is clear that the growing popular-ity of this class of methods stems largely from the ability of these approaches to provide an assessment of the accuracy of identification results that is virtually independent of the statistical approach used by the software that generated the result. This allows rigorous comparison of results derived from different software tools, as well as different instru-ments, sample preparations, and separation methods.

We present here a software tool that is designed to facili-tate result quality assessment by decoy methods. The tool provides an easy distillation of the yield of true positives at critical values of false-positive rates for both peptides and proteins. It also provides this information using two differ-ent types of false-positive rate calculations—the commonly used approach of assessing the error rate for all detections over a threshold, referred to as the “aggregate” rate, and also the “instantaneous” false-positive rate, which indicates the chance of error for detections with similar scores. The tool can be operated in a standalone mode to analyze the output from many search engines, or it can be operated as integrated into ProteinPilot software.

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Reproducibility of the Protein Forest digital Proteome Chip as a Protein Isoelectric Fractionation ToolA. Sin, J. Dasch, O. Kagan, P. Kandola, D. Artwell-Doucette, S. Haralampu, W. Skea, R. Garlick; Protein Forest, Inc., Waltham, MA, United States.

Protein fractionation is often necessary in mass spec-trometry (MS) proteomic studies to help detect low-abundance proteins. Protein Forest has developed the digital ProteomeChip (dPC) technology, which is a rapid, reproducible, and easy to use protein MS pre-fractionation tool.

The dPC consists of discrete pH gel features with pH intervals of 0.1 or smaller, which, when placed in an elec-tric field for 30 min, will each trap and collect proteins simultaneously from a complex mixture according to the proteins’ respective isoelectric points (pI). The resultant

protein fractions can then be tryptically digested for subse-quent MS identification analysis, transferred to traditional SDS-PAGE, or blotted to a membrane for antibody stain-ing. The individual pH features allow researchers to repro-ducibly extract protein fractions from a complex mixture for detailed analysis, such as in biomarker discovery and clinical assay applications.

We evaluated the isoelectric fractionation reproducibil-ity of the dPC technology using I125-labeled human growth hormone (hGH) as radioactive tracers spiked into a model complex protein mixture (E. coli lysate). We collected focused I125-hGH predominantly in the pH 5.2 to 5.4 frac-tions, and the average radioactive counts recovered were very reproducible, with a coefficient of variance of 16%. We also demonstrated the utility of a dPC in fractionat-ing E. coli lysates when coupled to subsequent LC/ESI-MS, SDS-PAGE, and immunoblotting analyses.

The dPC is a novel fractionation technology that pro-vides improved throughput and reproducibility compared to existing protein fractionation techniques. Our technol-ogy can be easily coupled to most existing downstream analysis techniques, allowing great research f lexibility. Finally, the robustness of the chip format, along with the digital nature, also makes the dPC amenable to customiza-tion and automation.

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Analysis of Glycoproteins in Human Serum by Means of Glyco-Specific Magnetic Bead Separation and LC-MALDI with Automated Glycopeptide DetectionK. Sparbier1, A. Asperger2, A. Resemann2, I. Kessler1, S. Koch1, T. Wenzel1, G. Shi3, G. Stein2, L. Vorwerg2, D. Suckau2, M. Kostrzewa1; 1Bruker Daltonics GmbH, Leipzig, Germany, 2Bruker Daltonics GmbH, Bremen, Germany, 3Bruker Daltonics, Billerica, MA, United States.

Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins and auto-mated analysis procedures for the detection, identification, and structural characterization of the corresponding pep-tide modification.

Selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically func-tionalized with lectins or boronic acids that bind to various structural motifs. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent iden-tification of low-abundant glycoproteins from serum is a challenging task.

Human serum was incubated with differentially func-tionalized magnetic micro-particles (lectins or boronic acids). Isolated proteins were released from the beads under acidic conditions, dried, and subsequently re-dissolved and digested with trypsin. The resulting complex mixture of peptides was subjected to LC-MALDI analysis. The respective glycoproteins were identified by direct MS/MS analysis and subsequent database searching. Intact glyco-peptides enriched by a second magnetic-bead purification



on peptide level were directly subjected to LC-MALDI analysis to get structural information about the glycan and peptide parts. A precondition to this novel approach was the discovery of certain consensus peak patterns in the MALDI-MS/MS spectra, allowing the automatic determi-nation of the peptide part and the glycosidic information of the glycopeptides supported by bioinformatics tools.

Applying this fast and simple approach, a high num-ber of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from experiments using magnetic ConA, LCA, WGA, jacalin, and boronic acid beads, respectively. The use of jacalin and boronic acid beads facilitates the enrichment of O-glycosidically modi-fied proteins. In contrast, ConA, WGA, and LCA spe-cifically bind N-glycosylated peptides and proteins. Few non-glycosylated proteins were identified, probably due to co-precipitation with glycosylated proteins.

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Proteomic Studies of Lipoxin A4–Induced Inhibition of Granulocyte-Macrophage Colony Stimulating Factor SignalingV. Starosta, K. Pazdrak, A. Kurosky; UTMB, Galveston, TX, United States.

Rationale: Granulocyte-macrophage colony stimulat-ing factor (GM-CSF) is one of the recognized contributors in the pathogenesis of lung diseases. It evokes a dramatic increase of phosphorylation events in the inflammatory cells after stimulation. Lipoxin A4 (LxA4) is a novel lipid mediator with putative proresolution properties that appear to function as inhibitory signal during the time course of inflammation. We investigated the influence of LxA4 on intracellular events during stimulation with GM-CSF.

Methods: 2D electrophoresis was used to resolve whole-cell proteome, subcellular compartments, and GM-CSF signaling-specific proteome, using biotinylated ligand immunoprecipitation and GM-CSF receptor immuno-precipitation as well. Overall protein phosphorylation was assessed on the gels with phospho-specific stain Pro-Q-Diamond and on the Western blot using anti-phospho-tyrosine immunostaining. Phosphorylation of ERK, SHP-2, and STAT-5 was revealed with specific antibodies. Protein interactions were assessed by co-immunoprecipitation. Cytokine profile was measured with multiplex assay.

Results: Pretreatment of the cells with LxA4 before GM-CSF stimulation led to the decrease of cytokine production as well as to changes in overall protein phosphorylation and tyrosine phosphorylation. Significant changes were also observed in phosphorylation of key kinases of GM-CSF signaling. Among signaling molecules affected by LxA4, STAT-5 transcription factor and SHP-2 phosphatase were detected. 2D electrophoresis showed that LxA4 pretreat-ment changed GM-CSF signaling in the proteome.

Conclusions: The inhibitory effect of Lipoxin A4 on GM-CSF-induced cellular function may indicate its poten-tial for downregulation of GM-CSF-driven inflammatory processes. We propose mechanisms of inhibition of GM-CSF action by Lipoxin A4 involving different cornerstones

of GM-CSF signaling, including protein phosphatases and transcription factor STAT-5.

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Protein Identification and Validation of Results in Workflows that Integrate over Various Instruments, Datasets, Search EnginesP. Hufnagel1, J. Glandorf1, G. Körting2, W. Jabs1, U. Schweiger-Hufnagel1, S. Hahner1, M. Lubeck1, D. Suckau1; 1Bruker Daltonik GmbH, Bremen, Germany, 2Protagen AG, Dortmund, Germany.

Analysis of complex proteomes often results in long pro-tein lists, but falls short in measuring the validity of iden-tification and quantification results on a greater number of proteins. Biological and technical replicates are manda-tory, as is the combination of the MS data from various workflows (gels, 1D-LC, 2D-LC), instruments (TOF/TOF, trap, qTOF or FTMS), and search engines.

We describe a database-driven study that combines two workflows, two mass spectrometers, and four search engines with protein identification following a decoy data-base strategy.

The sample was a tryptically digested lysate (10,000 cells) of a human colorectal cancer cell line. Data from two LC-MALDI-TOF/TOF runs and a 2D-LC-ESI-trap run using capillary and nano-LC columns were submitted to the proteomics software platform ProteinScape. The com-bined MALDI data and the ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen), and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined maximum false-positive rate of 5%. ProteinScape combined the data to one LC-MALDI and one LC-ESI dataset. The initial separate searches from the two combined datasets generated eight independent pep-tide lists. These were compiled into an integrated protein list using the ProteinExtractor algorithm.

An initial evaluation of the generated data led to the identification of approximately 1200 proteins. Result inte-gration on a peptide level allowed discrimination of protein isoforms that would not have been possible with a mere combination of protein lists.

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Evaluation of 36 MudPIT Replicate Datasets Using Normalized Spectral Counting and PLGEMS. K. Swanson1, N. Pavelka1, L. Florens1, T. Parmely1, J. W. Conaway1,2, R. C. Conaway1, M. P. Washburn1; 1Stowers Institute for Medical Research, Kansas City, MO, United States, 2Kansas University Medical Center, Kansas City, KS, United States.

Multidimensional protein identification technology (MudPIT) is a platform integrating multidimensional liq-uid chromatography and tandem mass spectrometry for protein profiling. As MudPIT becomes increasingly popu-lar, the full characterization of large datasets remains chal-



lenging. A total of 36 MudPIT technical replicates from two biological samples were acquired on XPs and LTQs ion-trap tandem mass spectrometers. This study evaluated the applicability of label-free quantitative proteomic informa-tion by fitting mean and standard deviation of normalized spectral abundance factors (NSAFs) to a power law global error model (PLGEM).

Protein fractions from affinity purifications of 293 cells expressing various Flag-tagged human Mediator subunits were enzymatically digested and analyzed by MudPIT. A total of 475 unique proteins were identified from 36 runs, resulting in a 36 × 475 matrix of relative abundance (NSAF values).

We observed a “goodness of fit” when PLGEM was applied to the entire 36 × 475 dataset, and subsets of the data containing only NSAF values from XP or from LTQ, indicating that NSAF datasets are compatible with this modeling scheme. Specifically, log-log plots of standard deviation vs. mean NSAFs revealed a linear dependence, with slopes ranging from 0.66 to 0.75 and an adjusted r2 equaled to 0.99. In addition, the residuals were found to be normally distributed across the entire dynamic range of average NSAF values. Similar ranges and distribution were also observed in Affymetrix GeneChip datasets.

This study demonstrates the utilities of NSAFs and PLGEM in statistically analyzing MudPIT datasets. With additional modeling using datasets from more complex samples or with “spiked in” experiments, this approach has a potential to become an integral data-mining tool for pro-teomics datasets acquired from one mass spectrometer as well as across multiple instrumentation platforms.

P187-S

Differential Proteome Analysis of the Cerebrospinal Fluid of Normal and Equine Protozoal Myeloencephalitis Affected Horses Using Two-Dimensional Fluorescence Difference Gel Electrophoresis and Mass SpectrometryD. E. Terry1, M. Levy2, I. Isaac3, N. Sharma3; 1Florida State University, Tallahassee, FL, United States, 2Purdue University, West Lafayette, IN, United States, 3Genomic Solutions, Inc., Ann Arbor, MI, United States.

Equine protozoal myeloencephalitis (EPM) is a com-mon and costly neurologic disease of horses caused by Sar-cocystis neurona, protozoa that parasitize the nervous system. Confirmation of the clinical diagnosis is a challenge for veterinarians because the current immunologic tests lack specificity. Multiplexing strategy approaches including two-dimensional fluorescence difference gel electrophoresis (2D DIGE) and mass spectrometry were employed to identify differential protein expression associated with EPM.

Cerebrospinal fluid (CSF) samples were collected from normal and EPM horses. EPM was diagnosed if neurologi-cal signs were present, Western blot against S. neurona on the CSF was positive, and other neurological diseases were excluded by ancillary tests and/or necropsy. CSF was col-lected under general anesthesia, at the atlanto-occipital site to avoid blood contamination. The CSF proteins were pre-cipitated, labeled with CyDye DIGE fluor dyes, and sepa-rated on 2D gels. The 2D gels were scanned with Typhoon,

and the digitized images were analyzed with DeCyder software. The differentially expressed proteins were excised with a ProPic II spot picker, in-gel trypsin digested, and the tryptic peptides were analyzed with the electrospray ioniza-tion mass spectrometer.

A number of differentially expressed proteins were identified in the various horse CSF, including serum albu-min, haptoglobin, prostaglandin-D synthase, and apolipo-proteins E and A-I. Various isoforms of serum albumin were up-regulated with EPM compared to the normal horse CSF. The above proteins, including the isoforms, may be associated with EPM, and the characterization of differen-tially expressed additional proteins could give insight into the pathogenesis and diagnosis of EPM.

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Long- and Mixed-Column Nanobore Chromatography for Complex Proteomic AnalysisC. J. Toher, A. W. Perala, C. J. Marshall-Waggett, G. A. Valaskovic; New Objective, Inc., Woburn, MA, United States.

Complex proteomic digests can require a combination of either multi-dimensional biphasic packed beds or longer reverse-phase beds (greater than 15 cm) to achieve ade-quate separation. Multi-dimensional and extended-length columns are time-consuming and expensive to produce; column failure of one section of the packed bed requires replacement of the entire column. An attractive alterna-tive to a single biphasic or long-bed column is based on efficient column-to-column coupling, as employed with conventional-bore high-performance liquid chromatogra-phy. Such connections in nanobore LC are enabled by cou-pling shorter-bed columns via optically clear high-pressure zero-dead-volume (ZDV) unions. This approach facilitates extended-column configurations and column swap-out during system maintenance. Using two clear ZDV unions to achieve flush connections to column bed termini, two conventional 10-cm-bed nanobore columns were coupled and connected to the bed terminus of a 10-cm nanobore column with integrally fritted tip. Analytical performance of this extended-bed column was then compared with a single 30-cm-bed nanobore column with integrally fritted tip. Chromatographic data from single- and extended-bed configurations were nearly identical, with negligible peak-tailing and resolution loss. The first section of the column is readily removed, replaced, and effectively integrates guard column performance into the analytical column bed.

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IMAC Enrichment of Phosphopeptides Coupled with 18O Labeling: Quantitative PhosphoproteomicsJ. L. Turner, J. J. Walters, J. D. Heutel, B. K. Radwanski, J. Boland, K. Ray, J. G. Dapron; Sigma-Aldrich Biotechnology, St. Louis, MO, United States.

The up- and down-regulation of protein expression has long been a focus of proteomics researchers and has given



rise to several mass spectrometry–based quantitation tech-niques. In the past decade, a great deal of attention has turned toward the subproteomes, particularly those rep-resenting post-translational modifications (PTMs), such as phosphorylation. Those PTMs often play key roles in signaling cascades, and affect a number of cellular pro-cesses; their study offers insight into intracellular function. We illustrate here phosphoproteome quantitation through the pairing of immobilized metal-affinity chromatography (IMAC) and 18O labeling, which allows for the determina-tion of modulated phosphorylated species in response to external stressors. Following application of varying biologi-cal stressors and isolation of each resultant proteome, the samples were proteolytically cleaved using immobilized trypsin. IMAC enrichment of phosphorylated peptides was accomplished using a nitrilotriacetic acid (NTA) analog bonded to silica, presenting Ga3+ as the chelated metal spe-cies. Immobilized trypsin was also employed for incorpo-ration of 18O into the sample for quantitative global label-ing. It was determined that IMAC enrichment utilizing this combination of chelate, substrate, and metal provided significant enrichment of phosphopeptides that afforded signal levels, which allowed for quantitative analysis of the phosphoproteome. The use of 18O labeling of the samples permitted a global approach to targeting the phospho-proteome concurrent with the non-phosphorylated com-ponent. Using MS analysis before and after IMAC-based phosphopeptide enrichment revealed a distinction in regu-lation of expression and modulation through phosphory-lation of the proteins. The coupling of phosphopeptide enrichment with global isotopic labeling provides a valu-able and enabling resource for the vastly under-explored area of quantitative phosphoproteomics.

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Statistical Significance of Differential Proteome ExpressionA. Wattenberg, P. Lutter, M. Blueggel; Protagen AG, Dortmund, Germany.

Differential proteome studies are a powerful tool for the analysis of differences between two sample states. A challenge encountered in any proteome study is the repro-ducibility of the sample preparation and data analysis. The significance analysis of the results and the extent to which changes can reliably be detected are affected by this.

We studied the changes of the proteome during cell dif-ferentiation using a combination of large format 2D gel electrophoresis, image analysis, and mass spectrometry.

The basis for any analysis is the reproducibility of the results and the study design. Firstly, the reproducibility of large-format 2D gel electrophoresis was shown. Two sam-ples of the same patient were analyzed using three replicate gels each. The spot quantitation of the two samples was found to be in good agreement. The relative mean standard deviation of the spot intensities within the replicate gels was 20% coefficient of variance. This allows us to analyze changes in the protein spot intensity that are smaller than a factor of two. The study design was optimized in order to account for technical and biological variation.

In the main study, 1800–2000 spots were quantified per gel. The large patient heterogeneity did not allow us to use a strict fold-change criterion for the selection of sig-nificantly changed spots between the two sample states. The variation of the spot intensity in one patient group was very much dependent on the nature of each individual protein. Therefore, a student’s t-test was employed to cal-culate the statistical significance for each spot. A total of 31 protein spots were found to be changed upon differentia-tion. Of these, 17 spots were unique for one of the samples, and another 14 spots were found to be highly significant (P = 99.9%). The effect of the Bonferroni correction and the false discovery rate is evaluated.

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A Comparison of nLC-ESI-MS/MS and nLC-MALDI-MS/MS for GeLC-Based Protein Identification and iTRAQ-Based Shotgun Quantitative ProteomicsY. Yang1, S. Zhang2, K. Howe1, T. W. Thannhauser1; 1USDA-ARS at Cornell University, Ithaca, NY, United States, 2Mass Spectrometry Facility, Cornell University, Ithaca, NY, United States.

The use of nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) in shotgun proteomics experiments and nano-liquid-chro-matography tandem mass spectrometry (GeLC-MS/MS) analysis is well accepted and routinely available in most proteomics laboratories. The same can not be said for nLC-MALDI MS/MS, which has yet to experience such wide-spread acceptance. This is not totally surprising, given the inability to monitor the separation performance in real time and the time-consuming processes of off-line data analysis and interpretation that are associated with MALDI. This is unfortunate, as the MALDI technology offers several criti-cal advantages over ESI that can be exploited to improve experimental outcomes. Here, we re-evaluate and demon-strate both the complementary nature of the two approaches and the synergism attainable through the employment of both ionization technologies to two common applications: protein identification in 1D gel bands and quantification of the dynamics of protein expression during ontogenesis.

Our results show that by using MALDI in addition to ESI in GeLC-MS/MS experiments, not only did the num-ber of proteins identified increase by an average of 15% but the average sequence coverage for proteins identified by both techniques increased by 175% over what it was when ESI was used alone. The size of the unique peptides identified by MALDI was, on average, 25% larger than the unique peptides identified by ESI. Furthermore, the higher resolution of the TOF/TOF instrument and the greater numbers of peptides detected using MALDI improved the sensitivity, accuracy, and precision of the data over what was obtained using only electrospray ionization in iTRAQ experiments carried out on a linear ion trap. These results demonstrate that the combination of nanoLC with two ionization technologies improves proteome coverage and quantitation in complex samples.



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On-Slide Chemical Modification as a Means to Improve Confidence in Protein Identifications Made by Peptide Mass FingerprintsX. Yang1, T. W. Thannhauser2; 1Cornell University, Ithaca, NY, United States, 2USDA-ARS, Ithaca, NY, United States.

The identification of proteins from large protein data-bases such as NCBInr using peptide mass fingerprints (PMFs) obtained on a MALDI-TOF mass spectrometer continues to be a challenge. A strategy that can be used to improve confidence in these identifications is to carry out some form of chemical modification reaction known to be specific for selected residues after the original analysis has been completed. The identification made by the original PMF is confirmed only if the mass shifts predicted by the putative identification are observed on reanalysis. To be most useful it would be necessary to carry out the chemi-cal modification “on slide” in such a way as to not com-promise the quality of the reanalysis. This requires that the chemical modification reagents be volatile or, at least, easy to remove. Oxidation with perfomic acid is a com-monly applied chemical modification that appears to meet these requirements. Here, we demonstrate the usefulness of on-slide peracid oxidation as a means to confirm PMF identifications.

P193-S

A Combined PTM Discovery Workflow as a Powerful Tool for Identification of Tyrosine Phosphorylation in GeLC-MS/MS AnalysisS. Zhang, C. Stearns, S. Baumgart, R. Cerione; Cornell University, Ithaca, NY, United States.

Tyrosine phosphorylation plays a pivotal role in signal transduction, which affects all aspects of cell life. Recently, mass spectrometry (MS) has been a powerful tool for iden-tification of tyrosine phosphorylation. Yet the MS approach is still far from being routine due mainly to low phosphory-lation stoichiometry at any given site.

The goal of this work was to identify the phosphotyro-sine (pY) residues on a human sorting nexin protein using nanoLC coupled with a triple-quadrupole linear ion-trap mass spectrometer. The initial identification was per-formed at enhanced survey MS (EMS)-driven information-dependent acquisition (IDA) mode. The subsequent valida-tion and further identification of low-stoichiometry pY sites were carried out using both precursor ion scan-triggered IDA and targeted multiple reaction monitoring (MRM)-triggered IDA. Compared to EMS-IDA analysis, the triple quadrupole–based precursor ion scan and MRM-driven IDA analyses provide significantly increased selectivity and sensitivity for detection of pY residues.

The immunoprecipitation samples from lysates of two transfected cell lines with Src kinase and ACK2 kinase were separated by SDS gel. The bands of interest (1–2 µg) were subjected to tryptic digestion/extraction. The extracts were split into three fractions for nanoLC-MS/MS oper-ated at EMS-IDA, precursor ion scan IDA, and targeted

MRM-IDA. The IDA files were used for database searches, and the spectra for identified pY peptides were manually inspected and validated.

The results showed that five pY residues were identi-fied by EMS-IDA and validated by the combined PTM discovery workflow for the Src kinase–catalyzed sample. The estimated stoichiometry of identified pY residues is from 3% to 25%. Furthermore, two pY sites were identi-fied by the PTM discovery approach for the ACK2 kinase–catalyzed sample. The results demonstrate that the triple quadrupole–based precursor ion scan and MRM-driven IDA provide excellent utilities for improved detection of phosphotyrosine residues without additional steps for enrichment of phosphopeptides.

quantitative proteomics

P194-M

Quantification of Protein Substrates From M. Tuberculosis Culture Supernatants Reveals Alternative Conserved Pathways for Virulence Factor SecretionM. M. Champion1, P. A. Digiuseppe2, J. S. Cox3; 1Applied Biosystems, Foster City, CA, United States, 2University Of California, San Francisco, CA, United States, 3University of California, San Francisco, CA, United States.

In addition to the general secretion pathway, Mycobac-terium tuberculosis utilizes an alternative secretion appara-tus called ESX-1, which is essential for control of host-cell responses to infection. Despite its importance in virulence, this system is poorly characterized and has few known sub-strates. There are five duplicate loci of the ESX-1 system in the genome, further increasing the difficulty in defining the nature of substrate secretion. Previous efforts to map the secreted proteome of this bacterium have been less suc-cessful because of the inability to differentiate true secreted substrates from those that are present due to lysis or sample preparation. We established and quantified secreted sub-strates from M. tuberculosis using a novel application of a known mass-spectrometric quantitative technique employ-ing isobaric mass tags and applied it to culture supernatants from wild-type M. tuberculosis and mutants deficient for components of ESX-1. Using this technique, we sought to identify ESX-1 substrates and determine whether dupli-cate Cfp-10 paralogs are secreted in an ESX-1-dependent manner. This has enabled us to definitively illustrate that most of the Cfp-10 paralogs are secreted in the absence of functional ESX-1, uncovering the presence of alternative secretion systems that have not been characterized. Also, we paradoxically found a quantitative increase in the secre-tion of substrates dependent upon Sec in ESX-1 mutants, suggesting cross-talk between general and alternative secre-tion systems. Further characterization of these substrates by mass-spectrometric quantification of dominant-nega-tive mutants gave further insight into the specificity and mechanism of ESX-1 substrate secretion.



P195-T

Reproducible Label-Free Quantitative Analysis of Low-Abundance Plasma Proteins using 3D Protein/Peptide Analysis Coupled with Protein Expression Analysis SoftwareE. Y. Chan1, C. L. Chepanoske1, S. Guedes1, A. D. Keller1, L. Weng1, L. A. Echan2, H. Tang2, D. W. Speicher2; 1Rosetta Biosoftware, Seattle, WA, United States, 2The Wistar Institute, Philadelphia, PA, United States.

Quantitative LC-MS/MS analyses of human plasma are predominantly challenged by the wide dynamic range of proteins and extensive biological sample varia-tion, which make systematic detection and quantitation of low-abundant proteins for biomarker discovery quite dif-ficult. In a proof-of-concept study, plasma samples were processed by alternative multi-dimensional workflows that first utilized immunodepletion of twenty abundant plasma proteins (ProteoPrep 20, Sigma). Multiple recom-binant proteins were spiked into depleted plasma samples at varying levels to determine detection limits of subsequent SDS-PAGE + LC-MS/MS vs. LC-MS/MS only. After SDS-PAGE, entire lanes were cut into uniform slices, and each slice was digested with trypsin. Digests were subsequently analyzed by nanocapillary reverse-phase chromatography directly coupled to an ESI-LTQ-FT mass spectrometer (Thermo) operating in data-dependent mode. In addi-tion, aliquots of each sample were either digested in solu-tion or as single in-gel digests after brief electrophoresis in an SDS gel, to evaluate advantages and complications of multi-fraction vs. single-fraction LC-MS/MS analysis of a proteome. Resultant LC-MS/MS data were analyzed in a blinded fashion using the Elucidator protein expression data-analysis system. Using the PeakTeller algorithm and statistical analysis tools, we were able to effectively organize the data to distinguish controls from spiked samples and estimate the relative abundances of spiked-in proteins in different samples. Detection limits, protein coverage, and reproducibility of the alternative workflows were compared. This study demonstrates that reproducible relative quanti-tation can be achieved in complex mixtures such as plasma, using a combination of abundant protein depletion fol-lowed by different workflow approaches and data analysis using the Elucidator system.

P196-S

Novel Software for Automated LC-MALDI and Relative Quantification of Multiplexed iTRAQ-Labeled Samples Using MALDI TOF/TOF InstrumentationJ. Connolly, R. Raso; Shimadzu Biotech, Manchester, United Kingdom.

The new generation of MALDI TOF/TOF instruments can be used to generate informative identification and rela-tive quantification data using iTRAQ chemistries. This presentation introduces a novel automated iTRAQ relative quantification software package for the AXIMA-TOF2 MALDI TOF/TOF mass spectrometer. The package uses

and extends onwards from existing LC-MALDI software. Interfacing of mass spectral data, relative quantification data, and Mascot search results in an interactive, visual correlation providing a highly practical end-user package.

The user interface has multi-level organization. The primary level shows Mascot database search hits along with an average quantification ratio for each protein along with significance scoring. The software correlates the peak intensity/area of the iTRAQ reporter ions to the protein identifications assigned by Mascot using MS/MS spectra in order to relatively quantify the protein components in labeled samples. Further interrogation of the individual peptide ratios may be found at the second visualization level along with the relevant spectra. The user interface allows input of iTRAQ kit–specific software correction factors, allowing duplex through to 8-plexed sample scenarios.

In proof-of-principle experiments combining iTRAQ relative quantification methodology with the AXIMA-TOF2, two equimolar aliquots of a six-protein mixture were derivatized with iTRAQ reagents 114 and 117 respectively. Relative quantification calculations were performed on the reporter ions and highlighted a 1:1 ratio of 114:117 with an error of ±15%, which is entirely consistent with the litera-ture. We have also established the necessity to acquire rela-tive intensity information for more than four peptide pairs in order to reliably quantify the parent protein.

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Automated Ion Selection and Method Building For MRM-Based Protein Validation And QuantificationE. Duchoslav, D. Cox, S. A. Tate; MDS Sciex, Concord, ON, Canada.

A number of successful multiple reaction monitoring (MRM)-directed MS/MS methods for protein character-ization and quantification have been published over the past couple of years. These methods have been created either by the generation of in silico MRM transitions or from taking discovery data generated by automated MS/MS analysis and formulating MRM transitions from this. However, such a workflow, where a vast amount of discov-ery data needs to be searched for idealized MS/MS to tar-get peptides, is difficult and time consuming. Automation of this would simplify the whole workflow, and allowing methods to be created smarter by utilizing the data of pep-tides that have been previously identified, provides a better chance of success.

In this poster we highlight one possible workflow for the processing of a number of database search results that were generated during proteomic analysis of a complex sample. Proteins identified as of potential interest in these results were then added to a MRM method-building list, and all MS/MS associated with those proteins were extracted from the general proteomic search results.

These MS/MS are then automatically processed to cre-ate instrument methods for MRM-directed MS/MS quan-tification. After analysis of the sample by these methods, the data are processed, both from a sequence identification and a chromatographic LC-MRM peak signal-to-noise



perspective. These data are associated with the original pro-teomic workflow data, allowing visualization of the results and construction of a method that can then undergo vali-dation.

P198-T

Label-Free Relative Quantitation of LC-MS Data Acquired from Trypsin-Digested Proteins in the Nucleus Accumbens of Rats Following Administration of Growth HormoneJ. Flensburg1, J. Samskog1, M. Le Grevès2; 1GE Healthcare, Uppsala, Sweden, 2Department of Neuroscience, Uppsala University, Uppsala, Sweden.

An important part of proteomics is to study changes in protein levels between samples from different cells or tissues where ideally all proteins present in the sample are monitored. There are two main methods that allow for both global scanning for significantly varying proteins, and targeted profiling of proteins of interest. One is based on 2D gel electrophoresis and image analysis of labeled proteins. The other method is based on LC-MS/MS analy-sis of either unlabeled peptides or peptides derived from isotopically labeled proteins or peptides. In this study, the non-labeling approach was used, involving DeCyder MS Differential Analysis Software (DeCyder MS), for auto-mated detection and relative quantitation of LC-MS data acquired from trypsin-digested proteins.

The study method aimed to identify and quantitate differentially regulated proteins following administration of recombinant human growth hormone (rhGH). Two groups of hypophysectomized male Sprague Dawley rats were daily subcutaneously injected during 8 d with rhGH and saline, respectively. The total soluble protein content of nucleus accumbens was extracted, digested with tryp-sin, and analyzed by 2D LC-MS/MS. In the first dimen-sion, the peptides were separated by micropreparative cat-ion exchange chromatography using an Ettan LC System. Collected fractions were analyzed in the second dimension using nanoscale RPC on Ettan NanoLC connected to an ion-trap mass spectrometer.

Acquired LC-MS data were evaluated using DeCyder MS, resulting in the detection of more than 5000 peptides, of which several were found to be significantly regulated. Sev-eral of these peptides could be correlated to proteins involved in synaptic plasticity as a response to growth factors.

P199-S

Assessing Signal to Noise in Quantitative ProteomicsD. B. Friedman, C. W. Whitwell, J. E. Loyd, B. Meyrick; Vanderbilt University Medical Center, Nashville, TN, United States.

The complex methodology used in many large-scale quantitative proteomics experiments often dictates an experimental design with small sample size and limited repetition. Variation in a complex dataset may hopefully arise from changes caused by the experimental perturba-

tion, but could also arise due to technical noise (poor sam-ple prep, run-to-run variation) and biological noise (normal differences between samples, especially present in clinical samples). Here, we apply principle component analysis (PCA) and unsupervised hierarchical clustering (HC) to the data generated from multi-variable difference gel elec-trophoresis (DIGE) experiments. This global perspective on multivariable datasets assesses whether the variation in the system describes the biological signal, rather than being derived from technical/biological noise whereby “sig-nificant” changes may arise stochastically. Although we use DIGE datasets as examples (due to the low technical noise), these issues are germane to all proteomics experimental platforms.

Examples will be shown from experiments containing samples from microorganisms, tissue culture, and clinical samples, where these tools were instrumental in demon-strating sample outliers, fouled samples, as well as varia-tion in sample preparation that overrides the variation from biological treatment (despite standard biological tests for sample validity). Experiments contained high-resolution datasets from multiple variables, with hundreds to thou-sands of protein forms monitored within each sample. Even with simple, two-condition experiments (e.g., WT vs. KO), these tests provide essential quality assurance and quality control.

One set of experiments focuses on familial pulmonary arterial hypertension associated with a mutation in the bone morphogenetic protein receptor 2. Affected individu-als carrying the mutation, familial obligates (carrying the mutation but asymptomatic), and a control set (married into the family) are all inter-compared. PCA and HC were used to assess the efficacy of drug treatment and the effect of inter-personal variation among the normal samples.

P200-M

Relative Quantification of Cerebral Spinal Fluid Proteins Utilizing Isobaric Tagging ReagentsS. Guertin1, B. Gomez-Mancilla2, M. Minkoff1, M. Willetts1, B. Williamson1, N. Guerreiro2; 1Applied Biosystems, Framingham, MA, United States, 2Novartis Institutes for BioMedical, Inc., Cambridge, MA, United States.

The development of isobaric tagging chemistries has permitted measurements in relative protein expression across several samples with a high degree of automation. Recently, a new set of isobaric reagents was developed that expanded the number of channels that can be processed simultaneously from four to eight. This doubling of the unique isobaric tags increases flexibility in the design of a multiplexing experiment, allowing the incorporation of a greater number of experimental samples, duplicates, or additional controls into a single pooled sample.

Described herein is a method to use these reagents to measure relative protein expression across several cerebral spinal fluid (CSF) samples. Eight control and diseased CSF samples were reduced, alkylated, and each labeled with one of eight isobaric tagging reagents. After labeling, all eight samples were combined into a single pool and fractionated



by cation exchange chromatography. The subsequent fractions were then analyzed by LC-MALDI on a 4800 MALDI TOF/TOF Analyzer. An analysis of the proteins identified in the CSF detailed several proteins that exhib-ited varying degrees of expression across the eight samples.

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ICPL Duplex and Triplex Technology for Quantitative Proteomics S. Hahner1, W. Jabs2, S. Brand1, S. Dikler3, G. Gounder4, K. Niehaus4, D. Suckau1; 1Bruker Daltonik GmbH, Bremen, Germany, 2Bruker Daltonik, Bremen, Germany, 3Bruker Daltonics, Billerica, MA, United States, 4University of Bielefeld, Bielefeld, Germany.

The isotope-coded protein label (ICPL) technology has shown to be efficient for comparative quantification of pro-teins. A major aspect of this chemistry is the labeling of all free amino groups on the protein level. This allows for the application of protein fraction technologies such as 2D gel electrophoresis, liquid chromatography, or free-flow elec-trophoresis of the labeled protein sample under retention of the dynamic range in the sample. In addition, labeled free amino groups, which are sufficiently available in proteins, result in a high number of representative labeled peptides after protein digest. This is of high importance because at least five labeled peptide pairs are required for a reliable determination of regulation in proteins.

We used ICPL labeling for the analysis of a complex pro-tein sample derived from the cell-wall proteome of Medicago truncatula. This plant belongs to the family of legumes that contribute about a third of the world’s protein requirements eaten by humans. Legumes are unique in their ability to fix atmospheric nitrogen through a symbiotic relationship with nitrogen-fixing bacteria Rhizobia. Thus, a compara-tive quantification study of extracted cell-wall proteins from M. truncatula is helpful for a better understanding of this particular plant-bacteria interaction.

The use of the newly developed ICPL triplex method will be shown, which allows for the comparative analysis of three proteomes. Furthermore, the data-handling aspects of multiplexed non-isobaric labeling was evaluated.

P202-S

Expanding the Capabilities of Peptide MRM-Based Assays in Plasma Using a Hybrid Triple-Quadrupole Linear Ion-Trap Mass SpectrometerC. Hunter; Applied Biosystems, Foster City, CA, United States.

As the study of protein biomarkers increases in importance, technical limitations to the detection of low-abundance proteins and high-throughput, high-precision quantitation remain to be overcome. The complexity and dynamic range of the plasma proteome makes the task of specific, quantitative detection even more challenging. Multiple reaction monitoring (MRM) capabilities of triple quadrupole MS systems have been explored as solutions to this challenge due to their well-known sensitivity and selec-

tivity for components in complex matrices such as plasma. Recently, a suite of >100 MRMs representing ~50 plasma protein markers were monitored quantitatively in a single assay using the MRM-based technique showing detection of proteins down to the level of L-selectin (~1µg/mL) with minimal sample preparation and no peptide or protein standards for most of the plasma protein markers.1

As more extensive candidate biomarker panels are being identified, MRM assays will need to be more rapidly developed to verify the expression changes of these proteins across larger clinical sample sets. To do this, the unique combination of triple-quadrupole and ion-trapping capa-bilities of the hybrid triple quadrupole–linear ion trap mass spectrometer have been utilized. A strategy for rapid MRM assay development for larger-scale profiling and qualifica-tion of biomarker candidates without having to first pre-pare synthetic peptide standards is currently being inves-tigated and involves a chemical labeling strategy to create global reference standards to enable quantitative compari-sons between clinical samples. Single assays consisting of ~500s of MRM transitions have been developed for this rapid qualification phase, facilitated by intelligent use of retention time windows during an LC analysis, while main-taining an optimum number of data points for improved precision of peak area and quantitative profiling. This pre-sentation will demonstrate the details of this workflow with human plasma examples.

reference

1. Anderson L, Hunter CL. Quantitative mass spectrometric mul-tiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics 2006;5(4):573–588.

P203-M

A Software Platform for the Validation of Quantitative Proteomics DataW. Jabs, S. Hahner, U. Schweiger-Hufnagel, M. Behrens, M. Lubeck, A. Asperger, H. Kaminski, L. Vorwerg, C. Bäßmann, D. Suckau; Bruker Daltonik GmbH, Bremen, Germany.

Methods based on isotope labeling and LC-MS/MS analysis are becoming a standard approach for the simul-taneous identification and quantification of proteins on the proteomic scale, as they provide high accuracy, pre-cision, sensitivity, speed, and compatibility with protein pre-fractionation. However, due to the high complexity of these experiments, a software platform is required that allows for efficient data acquisition, interpretation, and validation.

We describe such a validation platform for quantita-tive proteomics, which supports different types of mass spectrometers (e.g., MALDI-TOF/TOF, ESI-ion trap, -Q-OTOF, and -FTMS) and any label chemistry including multiplex labels. The software assists in the optimization of LC and MS instrument-specific parameters by providing 2D views on the whole LC-MS/MS dataset (SurveyViewer). The SurveyViewer facilitates tasks such as the fast assess-ment of the mass calibration or the fragmentation efficiency of isobaric labeling tags.



Protein regulations are derived from peptide regula-tion distributions using lognormal theory and median statistics. Median statistics is used in addition for the auto-mated elimination of outliers. Box and whisker plots and bar graphs assist in evaluating the quantitative results and providing the basis for the manual acceptance or rejection of individual quantitative data.

Direct links to sequence-annotated raw spectra of a par-ticular regulated protein or peptide with plots of the identi-fied amino acid sequence allow for efficient case study and curation of unexpected results, and the interactive valida-tion of uncertain results.

The described features of the software platform WARP-LC are exemplified in LC-MS/MS studies of protein mix-tures using ICPL, iTRAQ, and SILAC. Essential method-ological issues such as quantification based on median vs. average statistics and peak areas vs. peak intensities were evaluated. It appears that the monoisotopic peak intensities of de-isotoped isotopic clusters are preferable for quantifica-tion, as the average quantification errors are approximately 20% lower.

P204-T

Targeted Quantitative Protein Analysis in Human Plasma Using High-Resolution Multiple Selected Reaction Monitoring Assays on a Triple Quadruple Mass SpectrometerR. Kiyonami, K. Miller; Thermo Fisher Scientific, San Jose, CA, United States.

There is huge need for discovery and validation of novel biomarkers for early diagnoses of various diseases. Usually, a common endpoint for a biomarker discovery experiment is a list of putative marker proteins, and a reasonable next step will be to perform targeted quantitative measurements of these proteins in an expanded patient population to assess their validity as markers. Analytical accuracy and precision are required for unambiguous quantitative analysis of these targeted proteins from complex biological fluids, such as human plasma/serum. Wide dynamic range and high sensi-tivity are also critical for detecting low-abundance proteins from the complex samples.

One approach for this application is the use of tandem mass spectrometry to monitor a unique peptide (or peptides) from a protein of interest by a selected reaction monitoring (SRM) assay, or by simultaneous analysis of many peptides by a multiple selected reaction monitoring (mSRM) assay.This approach can be extended further to provide absolute quantitation of targeted proteins by incorporation of appro-priate stable isotope-labeled peptides as internal standards. While mSRM assays are sensitive for targeted peptides, in a complex matrix, such as human serum, assay selectiv-ity can become a major issue. It is often difficult to dif-ferentiate between the targeted peptide signal and matrix background, particularly when quantifying many very low abundance proteins. The unique high-resolution SRM (h-SRM) capability of the TSQ Quantum Ultra can help to significantly overcome this problem and increase mSRM assay specificity.

In this presentation, we demonstrate the TSQ Quan-tum Ultra mass spectrometer’s unparalleled capability for

highly sensitive and accurate multiple protein quantitation from human plasma by using high-resolution multiple reac-tion assays. Over 300 mSRM transition assays were devel-oped for detecting major proteins and known biomarkers simultaneously from human plasma by using both unit mass resolution and high-resolution on the Q1 quadruple.

P205-S

A Novel Toxicological LC MS/MS Application to Measure Biomarker Proteins in Largemouth BassC. J. Martyniuk, J. L. Blum, M. C. Dancel, S. M. Stevens, Jr., D. S. Barber, N. D. Denslow; University of Florida, Gainesville, FL, United States.

Biomarker discovery and application in toxicologi-cal studies requires rapid and accurate methods to assess environmental risk. The emerging field of proteomics now includes powerful analytical tools involving mass spec-trometry that can be utilized in toxicological studies. We are developing a focused proteomic technique using ESI- MS/MS to quantify the abundance of estrogen receptor (ER-alpha, ER-beta a, and ER-beta b) proteins in tissues of largemouth bass (LMB; Micropterus salmoides), a popular game fish that is used as a toxicological model. LMB ER sequences were cloned into expression vectors and recombi-nant proteins expressed for all three ER isoforms. Recom-binant LMB ER proteins were purified to use as standards. In silico trypsin digestions using ProteinProspector and MS-Digest of each LMB ER isoform were compared to identify peptides unique to each LMB ER. The peptide 406-LIFAQDLILDR-418 distinguishes LMB ER-alpha from the other two LMB ER isoforms, and analysis with purified LMB ER-alpha digests shows that this peptide ionizes consistently. For quantification, this peptide was synthesized with heavy isotopes (2nd leucine) to produce a peptide with a mass of 1322.6 Da, a difference of approxi-mately 6 Da when compared to the endogenous natural peptide mass of 1316.58 Da. A standard curve using the heavy isotope ER-alpha peptide showed linearity over five orders of magnitude and is detectable in the attomole range. Complex protein mixtures from the liver, gonad, and brain will be isolated using either whole-tissue protein or nuclear protein fractions for optimal compatibility with MS and ER peptide identification. In addition, Westerns blots are being developed for all three LMB ERs in parallel to deter-mine the most sensitive method of ER detection. These techniques are powerful toxicological tools to rapidly iden-tify changes in biomarker proteins such as ER isoforms. Research supported by NSERC PDF and Grants SBRP-P442E507375 and NIH-R01E5015449.



P206-M

Understanding the Metabolism of Amyloid-Beta in HumansK. G. Mawuenyega1, A. G. Wen1, K. Browning1, D. Holtzman1,2, R. Bateman1,2; 1Department of Neurology, Washington University School of Medicine, Saint Louis, MO, United States, 2Alzheimer’s Disease Research Center, Washinton University School of Medicine, Saint Louis, MO, United States.

The most common form of dementia is Alzheimer’s disease. According to the amyloid hypothesis, the disease is preceded by an accumulation of the amyloid-β (Aβ) pro-tein, which leads to downstream events including activa-tion of microglia, inflammation, synaptic dysfunction, and neuronal loss. The objective of this research is to address the physiology of Aβ in humans by measuring its in vivo metabolic rates.

A method was previous developed in our laboratory for measuring the in vivo synthesis and clearance rates of total Aβ within the cerebrospinal fluid (CSF) of normal healthy volunteers using a metabolic label of 13C6 leucine. After a spinal tap, the total Aβ peptide pool was isolated from the CSF by immunoprecipitation, followed by mass spectrome-try. As in selected reaction monitoring experiments, we use signals obtained from fragmentation of precursor peptide ions containing unlabeled and labeled leucine, but with a full scan tandem MS, instead of using precursor ions for quantitation as done in isotope-labeling experiments.

Total Aβ in human CSF was measured by ELISA meth-ods to be about 5000–20,000 pg/mL (~2 pmol total and 20 fmol 1% labeled). The metabolism of Aβ was measured by determining the percent 13C6 leucine labeling in Aβ in CSF samples taken every hour over a 36-h time course. The incorporated label was not detected until the fifth hour of label infusion, followed by an increase to a steady state at 20 to 24 h, then a decrease over the last 12 h.

In vivo metabolic protein labeling can be measured with high sensivtivity (low fmol labeled peptides), accuracy (R2 = 0.99), and reproducibility. These technical advances using common commercially available instruments may be applied to other proteins of interest and adapted to measur-ing total amounts of proteins or percent labeled proteins, for absolute and relative quantitation respectively.

P207-T

Protein Identification and Quantitative Analysis of Complex Mixtures Using a Peptide-Based Isobaric Mass Tagging TechnologyC. E. Parman1, H. Javali1, D. Wood1, H. Duewel2, C. Guerra1, B. Xie1, W. Patton1; 1PerkinElmer LAS, Waltham, MA, United States, 2Sigma-Aldrich Corporation, St. Louis, MO, United States.

A major goal in proteomics is to accurately measure changes in the relative abundance of large sets of proteins in complex biological systems. Isobaric mass tagging with ExacTag labels is a powerful platform for simultaneously analyzing relative protein levels from multiple complex samples. The technology provides a superior approach, combining high precision and reproducibility to accurately

determine the relative expression level of individual proteins in a complex mixture. A bioinformatics approach has been developed to enable fast and accurate protein identifica-tion and quantification through analyzing ExacTag-labeled peptides from MS/MS spectra. This approach combines popular protein identification programs such as Mascot or Sequest with an internally developed quantification soft-ware package. The results of the protein identification are used to predict labeled peptides, including possible post-translational modifications, which can be used for quan-tification. In parallel, each spectrum in the MS/MS data is scanned for ions coming from the mass tag labels. The package then searches the quantification results, matching the precursor mass to predicted peptides, and generates a list of quantified peptides for each protein.

This study focuses on accurately assessing the quan-titative consistency of the labeling technology, without introducing the confounding issue of biological variabil-ity. Complex samples were prepared consisting of different defined ratios of human serum and Escherichia coli bacterial cellular lysate. The protein mixtures were then labeled with different cysteine-reactive tags in order to compare protein expression levels among the different samples all at once using tandem mass spectrometry. The measured differ-ences in protein levels correlated well with the known input ratios of the two complex samples, reproducing changes in abundance of over 20-fold range. The described technol-ogy provides a powerful method for enhancing proteomic analysis by mass spectrometry.

P208-S

Top-Down Quantitative Proteomic Analysis Using a Highly Multiplexed Isobaric Mass Tagging StrategyW. F. Patton, B. Xie; PerkinElmer LAS, Waltham, MA, United States.

Proteomic analysis has proved key to determining drug mechanisms and assessing toxicological potential during preclinical screening studies. A major goal in proteomics is to accurately measure changes in the relative abundance of large sets of proteins in complex biological systems as a function of experimental parameters, such as drug dose or exposure time. Until recently, top-down quantitative pro-teomics has been restricted to 2D gel analyses or two-plex mass tagging. A new top-down approach based upon iso-baric mass tagging for highly multiplexing protein quanti-fication is presented, involving chemically tagging cysteine or lysine residues of intact proteins isolated from cells, tis-sues, or biological fluids. As many as ten labeled samples are then combined, fractionated, proteolytically digested, and analyzed by gel electrophoresis or LC-MS/MS. Pro-teins are identified using public domain search engines, such as Mascot (Matrix Science Ltd., London UK) and quantified using an in-house developed software package. During the fragmentation, the tag-labeled peptides generate a set of low-mass reporters that are unique to each sample. Measurement of the intensity of these reporters allows the relative quantification of the peptides, and consequently the proteins from which they originated. The capabilities of the approach are demonstrated by analysis of the HeLa cell nucleolar proteome after treatment with the metabolic



inhibitor actinomycin D for various time periods. A total of 542 proteins were qualitatively identified, and 232 of these proteins were then unambiguously quantified. The quan-tification data demonstrate that the nucleolar proteome changes significantly over time in response to differences in growth conditions, which is consistent with previous observations from several groups. The highly multiplexed and quantitative nature of the new technology should her-ald new opportunities to provide diagnostic and functional insights into the proteomics discovery process.

P209-M

Non-Isobaric Triplex-Labeling Strategies for ProteomicsU. Schweiger-Hufnagel1, M. Behrens1, S. Hahner1, E. Keidel2, D. Dosch2, S. Martin3, F. Lottspeich2, T. Halder4, D. Suckau1, C. Baessmann1; 1Bruker Daltonik GmbH, Bremen, Germany, 2Max-Planck Institute for Biochemistry, Martinsried, Germany, 3Bavarian Red Cross Blood Bank, Munich, Germany, 4Toplab GmbH, Martinsired, Germany.

A non-isobaric label chemistry is introduced that allows the triplexed quantification of proteomic samples based on the isotope-coded protein-labeling technology (ICPL). Amino groups of intact proteins were derivatized with three isotopically different nicotinoyl reagents (L: 12C6H4; M: 12C6D4; and H: 13C6H4) prior to protein separation and digestion. Here, we describe different approaches in respect of MS instruments and protein separation.

Protein samples with different complexity were labeled with ICPL. For one sample, the proteins were digested, sep-arated by CAP LC, and spotted on disposable targets for subsequent MALDI measurements. The other sample was separated on a 1D gel, excised, digested, and supplied to LC-MS/MS using a Qq orthogonal time-of-flight instru-ment.

Excellent results were obtained from the LC-MALDI and the Qq TOF approach with the three protein samples labeled differentially and measured together. The multi-plexed labeling allows us to reduce the experimental effort when proteomics experiments are run as replicates and when more than two different states are to be compared. A unique property of ICPL compared to other label chemis-tries is its compatibility with protein pre-fractionation, as the labeling reaction works on the undigested protein.

P210-T

Comparative Environmental Proteomics of Different Temperature Areas in Octopus Hot Spring, Yellowstone National Park.L. Steinke, R. Ramaley; University of Nebraska Medical Center, Omaha, NE, United States.

The microbial mats growing in the runoff channels of the hot springs of Yellowstone National Park (YNP) are a rich mix of bacterial, archaeal, and eukaryotic species. Mat samples were gathered from Octopus Hot Spring in 2005 and 2006.The samples were subjected to labeling with iTRAQ reagents followed by shotgun proteomics. Mascot

was used to query an in-house YNP database derived from the microbial portion of the NCBInr. It was expected that the majority of the proteins mapping to species with high temperature optima would be associated with the sample taken at a higher temperature, while those proteins from species with lower optima would be associated with the sample from lower temperature. Although Synechococcus is the most abundant microorganism in the mat community when abundance is measured by the percentage of DNA in a metagenomics sample, more peptides were identified from Roseiflexus sp. RS-1 than from any other organism. This discrepancy is most likely due to the sample being a mix of the large red and small green layers of the mat. Syn-echococcus resides only in the green layer. The large num-ber of distinct peptides from the Roseiflexus extracellular binding protein resulted in the highest Mowse score of the proteins identified. Eighty percent of the peptides associ-ated with the extracellular binding protein were isolated from the 58°C sample, while only 20% of this protein came from the 71°C sample. This trend continues throughout the housekeeping proteins quantified from Roseiflexus, as might be expected of a thermophile with a lower tempera-ture optimum. The remainder of the identified proteins showed the association with collection temperature that would be expected from the temperature optimums of the mciroorganisms from which they were extracted. The majority of the proteins identified in this experiment were housekeeping and structural proteins.

P211-S

Using 2D DIGE Instead of Conventional 2D Electrophoresis Dramatically Improves Statistical Validity of Differential Expression AnalysisE. Svensson, H. Nordvarg, S. Bourin, J. Buelles, L. Björkesten; GE Healthcare Bio-Sciences AB, Uppsala, Sweden.

Two-dimensional electrophoresis is an established method used to study differences in protein expression caused by, for example, a disease state or drug treatment. Conventional methods require the separation of one sample on each individual gel. This approach exposes the data to a high level of system variation, such as gel-to-gel variation caused by experimental factors. Quantification of protein differences can thus be uncertain and lead to false biological conclusions. Two alternatives to reduce this variation are (1) increase the number of replicates or (2) use the Ettan difference gel electrophoresis (DIGE) system. The Ettan DIGE system is a well-established technology in proteomics, which uses an internal standard for between-gel normalization. By pre-labeling samples prior to 2D elec-trophoresis with three spectrally resolvable CyDye DIGE Fluor dyes, electrophoretic co-migration of three protein samples on the same 2D gel is possible. This approach significantly reduces the number of replicate gels needed to ensure reproducibility and reliability of the differential expression analysis.

Here, we present results from a differential expression analysis experiment performed with Escherichia coli samples grown under different conditions. We demonstrate that by using DIGE and the DeCyder 2D co-detection algorithm,



the numbers of replicates are significantly reduced and the system variability is minimized compared to conventional electrophoresis with post-stained gels.

P212-M

High-Throughput Analysis of Protein Localization Dynamics by Mass SpectrometryD. J. L. Tan1, S. Hester1, A. Martinez-Arias2, K. Lilley1; 1Cambridge Centre for Proteomics, Cambridge, United Kingdom, 2Department of Genetics, Cambridge University, Cambridge, United Kingdom.

Cells are organized spatially and functionally into subcellular organelles, and a protein’s subcellular localiza-tion can provide a useful clue to its function. Additionally, changes in protein subcellular localization are involved in regulation of interactions, stability, and activity. For this reason, studying global changes in protein subcellular local-ization can provide useful insights into cellular functions.

We had previously developed localization of organelle proteins by isotope tagging (LOPIT), a high-throughput technique for protein localization to subcellular organelles. Organelles are partially separated by density gradient cen-trifugation. Gradient fractions are labeled with isotopic iTRAQ tags for quantitation by mass spectrometry. Pro-teins from the same organelle co-sediment exhibit similar distributions in the density gradient. By comparing distri-butions of unknown proteins to those of known organelle markers, subcellular localization can be assigned.

In this study, we extend the technique to look at pro-tein localization dynamics in the Drosophila melanogaster embryo during Wnt/Wingless signaling. This signaling pathway is involved in proliferation and differentiation, and several components are known to alter their localiza-tion in response to activation.

We have used LOPIT to simultaneously localize 851 Drosophila proteins to multiple organelles, including the plasma membrane, endoplasmic reticulum, and mito-chondria. The localizations were reproducible in indepen-dent experiments. Known organelle markers localized as expected, and novel proteins with previously unknown localizations were assigned putative localizations.

Repeating the analysis using embryos in which the Wingless pathway has been hyper-activated, we have iden-tified proteins that move in response to Wingless signaling, and are in the process of carrying out functional assays to dissect their role in the pathway.

P213-T

Comparison of Methods for Hemoglobin Variant Analysis that Provide a High Degree of AutomationS. A. Tate, C. Lock; MDS Sciex, Concord, ON, Canada.

Many of the available methods for the analysis of hemo-globin using mass spectrometry rely upon mathematical reconstruction of the data to generate information about the different isoforms. Although these produce the ability to speciate fine mass differences between various isoforms, they are computationally intensive and time consuming. These forms of mathematical reconstruction can lead to

artifacts in the reconstructed data, which may interfere with the accurate quantification of the different forms.

This poster presents data evaluating a number of dif-ferent methods for the determination of levels of modified hemoglobin. These include protein reconstruction, selected reaction monitoring (SRM) of protein charge states, and multireaction monitoring (MRM) evaluation of the spe-cific modified peptides. Using SRM screening, a number of different charge states have been monitored, which allows for shifts in the protein envelope due to differences in buf-fer composition or due to variations in the protein con-centration. This showed a low degree of specificity when compared to the use of protein reconstruction. The use of peptide MRM with subsequent MS/MS provided the best specificity, removing any ambiguity in the data. This also provided the best route for automation using a liquid chro-matography system and the Analyst Software 1.4.2 quan-tification module. This allows a fully automated workflow utilizing tools that provide features that assist users with good laboratory practices compliance.

P214-S

Identification and Quantitation of Isobarically Labeled Proteins: A Comparison of Separation StrategiesM. Willetts1, M. Minkoff1, P. Ulintz2, P. Andrews2, R. Marks3; 1Applied Biosystems, Framingham, MA, United States, 2Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, United States, 3Internal Medicine, University of Michigan, Ann Arbor, MI, United States.

Current protocols for protein quantitation using iTRAQ reagents utilize a tryptic peptide–labeling strategy. While there are many advantages to this workflow, it precludes the effective use of protein-level separation techniques. We compare two protein-level labeling workflows. The first is in-gel digestion after SDS-PAGE, and the second is solution-phase digestion after size-exclusion chromatogra-phy. Chromatography offers increased sample load relative to electrophoresis and thus potentially increased access to proteins of lower abundance.

The study was conducted on a human cell line (U937) infected with dengue virus, a mosquito-borne flavivirus of the Flaviviridae family. Intact protein samples were reduced, alkylated, and labeled at the lysine residues with one of the isobaric iTRAQ reagent tags, pooled, and then separated by size-exclusion chromatography. After separation, the frac-tions were digested with trypsin. These workflows resulted in larger peptides and reduced complexity, as the proteins are digested only at the arginine residues. The digests were then analyzed by LC-MALDI on a 4800 MALDI TOF/TOF Analyzer. Initial data show approximately 750 pro-teins identified using the gel-based workflow, with a simi-lar number of identifications from the size-exclusion data. The analysis will be focused on global protein expression changes as a result of infection in order to elicit information on how the virus competes with normal cellular mRNAs for the translational machinery of the cell.



P215-M

Protein Quantitation Using a Novel 8-plex Set of Isobaric Peptide LabelsB. Williamson; Applied Biosystem, framingham, MA, United States.

The advent of mass spectrometry–based tagging meth-ods, in particular amine-specific labeling reagents, has per-mitted relative expression measurements of large protein sets with a high degree of accuracy and reproducibility. The isobaric nature of the tags allows the protein samples to be pooled after labeling without increasing the complexity of the MS analysis. Identical peptides labeled with the differ-ent N-terminal reagents exhibit the same parent ion in MS. Upon MS/MS fragmention of the parent ion, unique signa-ture ions are generated, which indicate the source sample of each peptide and therefore the peptide’s relative abundance among the samples determined.

As tagging technology has become established and gained acceptance in relative protein expression analysis, there is a need to expand the scope of this technique to enable a higher degree of multiplexing. Described herein is a new set of reagents that doubles the number of states that can be compared from four to eight using the same robust N-hydroxysuccinimide chemistry and easy-to-use protocols as the original 4-plex amine-specific tags. Examples of the use of this reagent to quantitate eight states simultaneously will be shown and evaluated for label efficiency, fragmenta-tion efficiency, and precision and accuracy of quantitation.

P216-T

Beyond Differential In-Gel Electrophoresis: Top-down Multiplexed Analysis in Two-dimensional Gels Using an Isobaric Mass Tagging ApproachB. Xie, B. Hekking, H. Lisoukov, Y. Wang, C. Parman, W. F. Patton; PerkinElmer LAS, Waltham, MA, United States.

Large sets of biological samples are commonly encoun-tered in biomedical research, and a rigorous, reliable top-down quantification method for proteins from multiple samples is required. With differential in-gel electrophoresis (DIGE), Cy3 and Cy5 dyes are used to fluorescently label two different protein samples prior to running them on the same 2D gel. Often, Cy2 dye is used as an internal standard as well. This approach permits analysis of two to three samples under identical conditions, eliminating the need to register and match the images as in traditional 2D gels. Though conceptually seductive, problems plague DIGE, especially when attempting to perform complex quantitative proteomics studies. Since the DIGE images are generated with two to three different dyes of differing molar extinction coefficients, quantum yields, and physico-chemical properties, absolute quantification of small inten-sity differences is challenging. Also, DIGE is overly simplis-tic in its basic assumption that all information required in an experiment can be obtained from a single 2D gel using an easy control-vs.-perturbed state experimental design. Multi-point time-course experiments and drug dose-response experiments require many more than the two to three sample capacity of DIGE. An isobaric mass tagging

strategy is presented wherein seven different samples are simultaneously fractionated on a single 2D gel and protein abundances subsequently quantified by mass spectrometry. Workflows that replace or alternatively augment DIGE are described. Reproducibility of conventional DIGE vs. the isobaric mass tagging strategy demonstrates higher accu-racy quantification obtained with the latter approach.

P217-S

Relative Quantitation of iTRAQ-Labeled Proteins Using IRMPD on an LTQ FT UltraM. Zeller1, M. L. Blackburn2, M. Scigelova3, Y. Sun4, B. Delanghe1; 1Thermo Fisher Scientific, Bremen, Germany, 2Thermo Fisher Scientific, Somerset, NJ, United States, 3Thermo Fisher Scientific, Hemel Hempstead, United Kingdom, 4Procter and Gamble, Cincinnati, OH, United States.

An important and challenging aspect of proteomics is not only to identify all proteins from complex biological samples, but also to accurately measure their relative abun-dances.

To address this challenge many analytical methods have been developed, including several isotope labeling techniques: ICAT, SILAC, 18O, AQUA and iTRAQ. The data presented here describes a data dependent LC-MS/MS method where fragmentation using an infrared multi-photon decomposition (IRMPD) laser in the ion cyclotron resonance cell is employed to perform simultaneous protein identification and iTRAQ quantitation. The IRMPD frag-mentation technique produces spectra with rich fragmen-tation patterns, including fragments in the low m/z range. The resulting spectra also have high mass accuracy and resolution of the precursor and peptide fragments, includ-ing the iTRAQ reporter ions. Thus, the application of data-dependent LC-MS/MS using IRMPD fragmentation is ideally suited for confident identification and simultane-ous quantitation of complex protein digests.

recomBinant proteins

P218-M

Recombinant Protein Production in a Small Core FacilityP. S. Adams, M. W. LaMere, J. J. Hoffman; Trudeau Institute, Saranac Lake, NY, United States.

The age of proteomics and resulting advances in tech-nology have resulted in an increase in the number of requests for recombinant protein construction, produc-tion, and purification projects. One of the mainstays of the Molecular Biology Core Facility (MBCF) at Trudeau Institute is the production of Major Histocompatibil-ity (MHC) Class I and Class II multimeric fluorescently labeled protein-peptide complexes, which allow the track-ing of antigen-specific CD8+ and CD4+ T-cells using fluo-rescence-activated cell sorting (FACS) analysis. In addition, the MBCF has generated fusion proteins to be utilized



for immunization strategies and viral coat proteins from influenza and murine gamma herpes virus 68 for detecting epitope-specific antibodies. Another simple but extremely useful and money-saving protein production project is the production and purification of Taq enzyme for a laboratory that uses large quantities of Taq enzyme in a limiting dilu-tion assay for latent viral detection. Requests for milligram amounts of native, endotoxin-free proteins to be used for in vivo vaccination are increasing. The MBCF utilizes a vari-ety of expression systems, but primarily bacterial (using T7 promoter–based plasmids) and the insect cell–based Dro-sophila Expression System (DES) (Invitrogen). Recently, the Gateway system (Invitrogen) has been introduced into the MBCF to facilitate the whole procedure. Purification schemes are mainly based on size exclusion or affinity chro-matography using either protein A, Ni-NTA, or glutathi-one sepharose.

P219-T

Simultaneous and Systematic Evaluation of 96 Protein Refolding ConditionsP. A. Leland, J. L. Rane, J. G. Reich, A. Zilberman; EMD Biosciences, Inc., Madison, WI, United States.

Proteomics requires large amounts of highly pure and correctly folded protein. This need is often met using het-erologous expression systems such as Escherichia coli. Over-expression of foreign proteins in E. coli commonly results in the formation of inclusion bodies—dense, insoluble aggre-gates of misfolded protein. Though commonly viewed as detrimental, inclusion bodies are easily purified, resistant to proteolysis, and can be solubilized with chaotropic agents. Defining conditions that promote refolding of a chemically solubilized target protein into its native confor-mation, however, is largely empirical. The chances of iden-tifying an optimal refolding condition can be increased by simultaneously and systematically evaluating a large num-ber of refolding conditions. To meet this need, we have developed the iFOLD Protein Refolding systems, a col-lection of 96-well plate-based refolding screens. System 1 uses N-lauroylsarcosine, a chaotropic anionic detergent, to denature the inclusion bodies, while System 2 uses either guanidine hydrochloride or urea as the denaturant. With both systems, proteins are refolded by rapid dilution of the denatured inclusion bodies into the 96-well refolding plate. The plate contains 92 (System 1) or 95 (System 2) unique protein refolding solutions, each comprised of buffers, salts, redox couples, and protein refolding additives. Using the systems, we have successfully identified high-yielding refolding conditions for a green fluorescent fusion protein, a mammalian endopeptidase, a matrix metallo-protease, and a viral protease. Refolding was measured using spec-trophotometric, f luorescent, and target-specific activity assays. High-yielding refolding conditions identified by the screens have been scaled 10,000-fold from 50 µg to 500 mg. Significantly, all steps of the iFOLD systems are equally compatible with manual use and high-throughput automated liquid-handling systems.

P220-S

Dual-Purpose Insect Cell Expression Vector for Transient Transfection and Baculovirus GenerationK. Loomis, C. Rockwell, H. Sternard, R. Novy; EMD Biosciences, Madison, WI, United States.

Baculovirus-mediated expression has proven to be a robust method of generating recombinant proteins from insect cells. However, generating baculovirus recombinants using traditional techniques is time consuming and tedious. To accelerate the process of insect cell expression, EMD developed a rapid transient transfection-based approach, the InsectDirect System. This approach is well suited for the rapid generation of small to moderate amounts of recom-binant protein. For situations that demand the baculovirus approach, we introduced a technology for rapid baculovirus production, BacMagic DNA. To optimize, simplify, and consolidate both systems. we created a single expression vector compatible with both approaches, pIEx/Bac.

To direct expression by transient transfection, pIEx/Bac expression vectors feature the homologous region 5 (hr5) enhancer and the immediate early 1 (ie1) promoter from Autographa californica nuclear polyhedrosis virus (AcNPV), a promoter/enhancer combination that uses endogenous insect-cell transcriptional machinery. To direct expression during baculovirus infection, the above promoter/enhancer combination is active during the early stage of baculovirus infection, and an additional p10 promoter directs expres-sion in the late/very late phases. We also created additional vectors featuring alternative combinations of baculovirus-derived promoters and enhancers to verify that our par-ticular combination of enhancer and promoter elements was optimal. Using the Radiance Ek/LIC cloning method, the reporter enzyme Renilla luciferase (Rluc) was cloned into all variants to allow comparisons of relative expression strength. The plasmid that gave the best overall perfor-mance in both systems was chosen as our new dual-purpose expression plasmid, pIEx/Bac. To further test the vector, we cloned additional inserts encoding differing classes of pro-teins, including an importin, a phosphatase, and kinases. The results demonstrate the utility of the pIEx/Bac vector for streamlined expression in insect cells.

P221-M

Autoinduction of Protein Expression for NMR Structural StudiesJ. G. Reich1, P. A. Leland1, J. L. Rane1, P. G. Blommel2, K. Becker2, B. T. Burns2, J. Song2, J. L. Markley2, B. G. Fox2; 1EMD Biosciences, Madison, WI, United States, 2Center for Eukaryotic Structural Genomics and Department of Biochemistry University of Wisconsin-Madison, Madison, WI, United States.

The development of the autoinduction process by Studier has greatly streamlined bacterial protein expres-sion. In autoinduction media, bacterial expression sys-tems controlled by lac promoters (such the pET system) grow uninduced to a high cell density and then sponta-neously induce high-level protein expression without the need to monitor cell density or add isopropyl β-D-1-thio-galactopyranoside (IPTG). These media offer significant



increases in target protein yield compared to conventional IPTG induction. Autoinduction media are ideally suited for high-throughput, parallel analysis of protein expres-sion, solubility screening, and purification from multiple expression clones. Here we describe a new autoinduction medium that enables production of [U-15N] and [U-13C, U-15N]-labeled proteins for NMR structural studies. We expressed and purified four [U-15N]-labeled target proteins and one [U-15N, U-13C]-labeled protein from Arabidopsis clones using this medium. NMR analysis of each labeled protein returned high-quality HSQC spectra with greater than 95% isotope incorporation, as calculated from mass-spectrometric data. The isotopic labeling medium (Over-night Express NMR) can generate labeled proteins from colonies in two work days, and will soon be commercially offered as pre-sterilized, ready to use solutions.

P222-T

Increasing the Ease and Speed of Eukaryotic Protein Expression: A Cell-Free In Vitro Translation System Based on Sf Insect Cell ExtractsJ. Smith1, F. Schaefer1, A. Zacharias1, N. Brinker-Krieger1, U. von Groll1, S. Kubick2, K. Steinert1; 1QIAGEN, Hilden, Germany, 2RiNA GmbH, Berlin, Germany.

We set out to develop an easy-to-use system for in vitro synthesis of recombinant eukaryotic proteins containing post-translational modifications. A wide range of eukary-otic proteins require such modifications (e.g., phosphoryla-tion, glycosylation, signal peptide cleavage) for correct fold-ing or to display full functional activity.

To optimize expression, a number of expression con-structs were generated using polymerase chain reaction (PCR). These constructs encoded proteins carrying com-binations of C- or N-terminal His- or Strep- affinity tags. Promoters added during the PCR enabled the efficiency of expression and proportion of soluble protein delivered by each construct to be tested directly in small-scale synthe-sis reactions. Once the makeup of the optimal construct was determined, it was cloned into an expression plasmid for larger-scale syntheses. A range of proteins—including kinases, membrane proteins, and transcription factors— were expressed to test the robustness and applicability of the method. Optimized Spodoptera frugiperda cell lysates, which contain all the cellular machinery required for post-translational modifications, were used for expression. The presence of post-translational modifications in synthesized proteins was demonstrated using epitope-specific antibod-ies (phosphoproteins), enzymatic treatment (glycoproteins), and activity assays (luciferase).

The ability to use PCR products in small-scale expres-sion screening reactions speeded up the entire process of construct optimization considerably. It was possible to synthesize a wide range of post-translationally modified proteins in high yields (up to 40 µg/mL reaction), signifi-cantly higher than those obtained in a comparison with a rabbit-reticulocyte lysate-based system.

This system offers a range of benefits for eukaryotic protein expression, including simplified expression screen-

ing, a fast procedure, robust and high-yield expression, and no requirement for specialized equipment.

sequence management and analysis

P223-S

A Tale of Two Technologies: From Slab Gel to Capillary, Updating the Biomolecular Core Laboratory Genotyping UnitJ. Holbrook, C. Agresta, D. L. Stabley, K. Sol-Church; A.I.duPont Hospital for Children, Wilmington, DE, United States.

“It was the best of times, it was the worst of times” (C. Dickens: A Tale of Two Cities)

The Biomolecular Core Laboratory (BCL) provides essential services in molecular biology and genetics to the research and clinical staff at the Alfred I. duPont Hospital for Children and affiliates.

In 2004, the Biomedical Research Department was awarded a Center for Biomedical Research Excellence grant from the National Institutes of Health for the establish-ment of a new center for pediatric research. Funds were available for BCL to upgrade existing DNA sequencing and genotyping services and establish new high-throughput gene expression services. There were many challenges encountered when optimized assays were moved from the ABI Prism 377 DNA Sequencer to the ABI Prism 3130xl 16-capillary Genetic Analyzer.

Customers experienced difficulties when using sequenc-ing protocols that were optimized for the slab gel system, on the new capillary system. The wide variety of genetic tests offered for molecular diagnostics had to be re-evaluated and optimized to fit the enhanced capabilities of the new platform. Even the chromatogram printouts looked differ-ent, and it took our customers (and ourselves) some time to adjust the settings to fit customers’ needs.

In this poster, we will outline the steps involved in updating sequencing technology from slab-gel to capillary electrophoreses and present how, through multiple rounds of trial and error, customers and core staff were able to re-evaluate existing assays and identify several impor-tant factors (DNA purity, BigDye dilution factor, buffer conditions, removal of dye blobs, software settings) that contribute to sequencing success. We will also report on a customer survey that was conducted to evaluate core performance, identify areas needing improvement, and highlight the strengths of our new programs and the steps taken to improve customer satisfaction, data quality, and cost effectiveness.



P224-M

Management Systems and Storage of Laboratory Information at Laval University Hospital Research Center Genomic Sequencing and Genotyping PlatformM. A. Rodrigue, É. Fortier, A. Gareau Pagé, P. Beaulieu, V. Raymond; Laval University Hospital Research Center (CRCHUL) Genomic Sequencing and Genotyping Platform, Québec City, PQ, Canada.

Between November 2005 and October 2006, a total of 106 academic and corporate researchers requested the services of the CRCHUL Genomic Sequencing and Geno-typing Platform. All procedures are performed by only one manager and two research assistants. During this one-year period, we sequenced 205,000 DNA samples using only one ABI 3730xl DNA analyzer. We also genotyped 8200 samples using one ABI 3100 sequencer. The maximum delivery time was 48 h for all projects of less than 384 samples. These samples comprised 80% of all our orders. The management and integration of all these samples were conducted on a computer system that we developed in house. This laboratory information management system has significantly reduced processing and analysis time and concurrently increased production capacity. All data are stored on our Oracle 10G database installed on a UNIX server, which is connected to an interface application server module to increase security. The Web interfaces were devel-oped in PL/SQL, which facilitates quick consultations, additions, and modifications. Our customers can thus eas-ily submit their samples at www.sequences.crchul.ulaval.ca. All laboratory procedures are tracked and managed by two research assistants through our laboratory information management system (LIMS). This LIMS also allows the customers to follow the progress of their samples on line. Once the sequencing process is completed, all samples are analyzed by our personnel, a descriptive note is added to every sample, and an electronic notification is sent to the customer. Results are retrieved from our database by down-loading a compressed file. Production of monthly finan-cial statements is incorporated into our computer system, reducing administrative time and expenses with respect to management. In the near future, we will be integrating our most recent SNP genotyping service using the Sequenom MassArray into our information management system.

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Influence of Sample-Loading Solutions on Sequencing Performance and Loss of Resolution on the Applied Biosystems 3730 DNA AnalyzerJ. A. Fisher, S. R. Berosik, K. B. Lee, S. Santhanam; Applied Biosystems, Foster City, CA, United States.

We have investigated the effects of sample-loading solu-tions on sequencing performance on the Applied Biosys-

tems 3730 DNA Analyzer, and in particular their effects on the decline of sequencing performance, or loss of resolution. We compared sequencing performance of samples resus-pended in water, 0.1 mM EDTA, or deionized formamide. Our results show that samples resuspended in water give poorer overall performance in terms of average number of Q20 bases per sample and in the amount of variability in Q20 scores, compared to samples in formamide or 0.1 mM EDTA. Over the course of 300 or more runs, samples in water had a significantly greater tendency towards gradual loss of resolution, while EDTA and formamide samples maintained steady high performance. We will present additional data dealing with relative advantages and disad-vantages of the three solutions, such as signal strength and sample stability, but we conclude that EDTA and deionized formamide are the preferred loading solutions for sequenc-ing applications.

P226-S

Direct Sequencing Quality Control: A Novel Software Approach to Reducing Variant Review Time and LaborE. Vennemeyer, S. Jankowski, K. Hunkapiller; Applied Biosystems, Foster City, CA, United States.

With the completion of the Human Genome Project, the shift from de novo sequencing to direct sequencing (resequencing) has created the need for more accurate vari-ant detection for medical research and clinical diagnostics. The bottleneck in the workflow (from DNA extraction to result data analysis), which has been cited as taking up to 70% of researchers’ time per project, is the manual review of individual nucleotide bases. This review has been required due to the necessity of having confidence in the variant result.

Increasing confidence can come from applying diligent quality-control metrics, including use of quality values for DNA trace value and confidence values for variant validity. Based on Applied Biosystems experience, this system will filter out low-quality data. The software will then direct users to review only low-confidence variants.

A flexible workflow-based system is being built to enable researchers to obtain their high-confidence results in less time. Methods for filtering low-quality data based on optimal settings and quality visualization tools will be integrated into the system along with simpler variant review and reporting tools to allow researchers to quickly analyze their data.

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P133-S An Improved Protocol of Coupling Synthetic Peptides to KLH for Antibody Production Using DMF

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