2.2 2.3 Free-Flow Electrophoresis: A Highly Quantitating Molecular

2.2Free-Flow Electrophoresis: A HighlyVersatile Fractionation Method for ProteinAnalysis and ProteomicsChristoph Eckerskorn

Tecan Munich GmbH, Kirchheim, Germany

Free Flow Electrophoresis (FFE) is a highly versatile technology for theseparation of a wide variety of charged analytes like low-molecular weightorganic compounds, peptides, proteins, protein complexes, membranes,organelles, and whole cells in aqueous media under native and denaturingconditions. The analyte is injected into a thin, laminar separation buffer film(defining the electrophoretic separation mode like zone electrophoreses,IEF, ITP) and deflected by an electric field perpendicular to the flowdirection. The absence of any kind of matrix is directly linked to highsample recoveries of more than 95% and fast fractionation times of lessthan 20 min. This predestines this technology to overcome one of thebiggest challenges in proteome research: the reduction of the complexityof the samples by fractionation. Furthermore, the enrichment allows thevisualization of less abundant proteins for subsequent separation by two-dimensional electrophoresis or LC-MS analysis.

A raw mitochondrial preparation from Saccharomyces cerevisiae ob-tained by differential centrifugation was purified by FFE. 2D-electrophore-sis and MALDI-MS analysis of this clearly demonstrated the strong pres-ence of protein fragments and proteins of non-mitochondrial origin.Strikingly, the 2D-patterns of FFE purified mitochondria yielded only fulllength protein species of mitochondrial origin as demonstrated by morethan 400 PMF identifications. Moreover, it was possible to enrich andidentify low abundance proteins, which were not present on the 2D-mapsof the raw mitochondrial preparation.

FFE fractions from liver lysates separated on consecutive narrow pH-gradients resulted in protein patterns of more than 15,000 protein spots. Itwas observed that a series of proteins showed a significantly higherabundance compared to separations of non fractionated protein mixtures,especially in the high molecular mass range. The visualization of proteinsin extreme pH-ranges could be partially enhanced, either on dedicated pHstrips or, if below the current possibilities of IEF, on SDS-PAGE. In suchcases, several hundred proteins with a pI � 3 could be detected. Theseparation of a large number of membrane proteins and trans-membraneproteins was achieved by FFE-prefractionation of isolated membranessolubilized in non-ionic detergents followed by SDS-PAGE.

Conclusion: The fractionation of cell organelles or complex proteinmixtures by FFE in combination with PAGE allows access to less abundantproteins species as well as to proteins with extreme physical-chemicalproperties like high mass range, hydrophobicity and extreme IEPs.

2.3Quantitating Molecular InteractionsDavid Myszka

University of Utah, Salt Lake City, UT

Characterizing the time component of protein interactions is essential forrecreating molecular pathways. Fortunately, today we have access tooptical biosensors that allow us to study the interactions of nearly anymolecular complex in real time without the need for labeling. These sur-face plasmon resonance (SPR)-based sensors are playing major roles indefining molecular activity in terms of the kinetic rate constants of binding.Evolving applications such as the study of membrane-associated recep-tors and small molecule detection, along with next generation array-basedSPR biosensors capable of monitoring up to 400 reactions simultaneously,are expanding the ways we apply biosensor technology. And, given theexplosive interest in characterizing functional aspects of the proteome,biosensors have arrived just in the nick of time.

A.1Peroxisomal Proteomics, a New Tool toStudy Peroxisomal DisordersJia Mi and Susana Cristobal

Uppsala University, Uppsala, Sweden

Mass spectroscopy remains one of the most powerful tools for the inves-tigation of genetic metabolic diseases. Peroxisomal biogenesis disorders(PBDs) arise from genetic defects in the assembly of peroxisomes. It is aheterogenous group of genetic diseases that present a challenge to childneurologists because of their prominent and devasting neurological symp-toms. We use peroxisomal proteomics to generate the basis scaffold tounderstand which metabolic functions are defective and to explore ther-apies. Peroxisomes from mouse tissues: liver, kidney and brain wereobtained by a modified immunoisolation method developed in our labo-ratory. The method is based on the interaction of specific antibodies(PMP-70) bound to a solid support. Peroxisomal matrix proteins from wildtype mouse tissues have been separated by two-dimensional gel electro-phoresis with denaturing isoelectric focusing in the first dimension (pI3–10, 4–7 and 6–11) and SDS-PAGE (10%) in the second dimension. Weprocessed several 2DE maps to obtain a master image for each of wildtype tissues. Finally, we have currently achieved the identification of 50proteins from a total of 70 isolated spots using MALDI-TOF MS. Proteinssuccessfully identified are involved in the main peroxisomal biochemicalpathways, such as �-oxidation and amino acid and purine metabolism.Experiments using fluorescence 2-D difference electrophoresis technique(DIGE) with peroxisomes from a knockout mouse model for these disor-ders are on course. We expect to identify which peroxisomal enzymes arepresent in abnormal concentration during the course of the disease andwe will analyze which metabolic pathways have been disrupted.

Mass Spectrometry in the Health & Life Sciences

S20 Molecular & Cellular Proteomics 2.7

by guest on April 12, 2019

http://ww

w.m

cponline.org/D

ownloaded from

http://www.mcponline.org/

A.2Combining Metabolomics and Proteomicsin Plant Systems Using GC/TOF andMultidimensional LC/MSWolfram Weckwerth

Max Planck Institute of Molecular Plant Physiology, Potsdam,Germany

At a systems level, gene function is regarded as being dependent on thedevelopmental stage, environmental conditions, and expression levels ofother genes, resulting in dynamic changes in transcript, protein and me-tabolite profiles. Thus, the next stage of understanding necessitates thatbiological tissues be described in depth for different levels, i.e. not only atthe level of transcripts or protein expression, but also at the metabolitelevel and the dynamic interaction of different gene products.

A novel extraction protocol is described with which metabolites, pro-teins, and RNA are sequentially extracted from the same sample, therebyproviding a convenient procedure for the analysis of replicates. Detectionof metabolites, proteins, and RNA bands from a single Arabidopsis thali-ana leaf sample was validated by analysis with GC/TOF/MS, two-dimen-sional LC/MS for proteins, and northern blot analysis for RNA.

652 metabolites, 297 proteins, and clear RNA bands were detectedfrom a single Arabidopsis thaliana leaf sample by GC/TOF/MS analysis formetabolites, two-dimensional LC/MS for proteins, and northern blot anal-ysis for RNA. A subset of abundant protein and metabolite data of repli-cate analysis from different Arabidopsis varieties were merged to form anintegrated dataset, allowing both classification of different genotypes andthe unbiased analysis of the hierarchical organization of proteins andmetabolites within a real biochemical network. A theoretical model forinterpretation of the metabolomic network is discussed.

A.3Probing Ffh-FtsY Complex Interface UsingChemical Cross-linking and MassSpectrometryFeixia Chu and Alma L. Burlingame

Mass Spectrometry Facility, Department of PharmaceuticalChemistry, University of California, San Francisco, CA

Among the methods to study the nature and extent of the protein inter-action interface, chemical cross-linking followed by identification of thecross-linked peptides by mass spectrometry has proved especially usefulin dynamic and complex systems when crystallization of the protein com-plex is difficult to achieve. Ffh, the protein subunit of the signal recognitionparticle (SRP) in bacteria, and the SRP receptor, FtsY, play a critical rolein delivering nascent membrane and secretory proteins to the bacterialplasma membrane (1). At the very heart of this protein targeting reaction isthe recognition between Ffh and FtsY. The x-ray crystal structures of bothFfh and FtsY have been solved (2, 3), however, the Ffh-FtsY complex hasso far eluded crystallographic analysis.

We have previously reported an integrated generic method to probeprotein-protein interaction interfaces, using chemical cross-linking, differ-ential nano-LC-MS, computational prediction for cross-linked peptidesand nano-LC-MS-MS for the establishment of the cross-links (4). Usingthe concepts of Zscore (5) to deisotope complex LC-MS runs, we havedeveloped a program to differentiate related LC-MS runs. To this end, wehave investigated various cross-linking conditions and optimized thecross-linking yield of the Ffh-FtsY complex using the homobifunctionalcross-linker DSS (disuccinimidyl suberate). The cross-linker treated mono-mers of Ffh and FtsY, were combined and used as a negative control. TheLC-MS runs of both the cross-linked Ffh-FtsY complex and negativecontrol tryptic digestion mixtures were thoroughly compared by our pro-gram in searching for cross-linked peptides. Two inter-molecular cross-linked peptides were identified, and CID (collision induced dissociation)spectra of these two peptides were obtained to establish the cross-linkson the Ffh-FtsY dimer interface. The cross-linked residues are located inthe hinge region between the N domain and the G domain of eachindividual protein, in agreement with mutation studies on FtsY, indicatingthat this region may be important for the conformational changes thataccompany Ffh-FtsY complex formation. A model of the Ffh-FtsY com-plex will be discussed based on the crystal structure of each individualprotein as well as the results on the cross-linking study of the complex.This work was supported by NIH NCRR BRTP RR01614 (A.L.B).

1. Keenan, R. J., et al. (2001) Annu. Rev. Biochem. 70, 755–775.2. Freymann, D. M., et al. (1997) Nature 385, 361–364.3. Montoya, G., et al. (1997) Protein 28, 285–288.4. Chu, F., et al. (2002) 50th ASMS Conference on Mass Spectrometry

and Allied Topics, June 2–6, 2002, Orlando.5. Zhang, Z., et al. (1998) J. Am. Soc. Mass Spectrom. 9, 225–33.


Molecular & Cellular Proteomics 2.7 S21


http://ww

w.m

cponline.org/D

ownloaded from


A.4Enrichment and Identification of WEHI 231Phosphoproteins Using IMAC andLC-MS/MSHongjun Shu, She Chen, Robert Hsueh,and Deirdre Brekken

University of Texas Southwestern Medical Center, Dallas, TX

A goal of the Alliance for Cellular Signaling (AfCS) Protein ChemistryLaboratory is the identification of phosphoproteins in mouse B lympho-cytes (B cells). In order to identify phosphoproteins on a proteome-widebasis, WEHI-231 cells were treated with calyculin A, a serine/threoninephosphatase inhibitor, or left untreated. Proteins were extracted fromwhole cell lysates and treated with trypsin to generate peptides. Phos-phorylated peptides were enriched using immobilized metal affinity chro-matography (IMAC) and identified by liquid chromatography-tandem massspectrometry (LC-MS/MS). A total of 120 proteins were identified usingthese methods. Forty-two of these proteins were previously reported to bephosphorylated, 14 of which are on the AfCS molecule list. The list alsoincludes 24 proteins that appear to be completely novel. A total of 213phosphorylation sites were identified within these proteins. A single siteof tyrosine phoshorylation was identified, presumably due to the verylow abundance of this modification relative to serine/threoninephosphorylation.

A.5Quantifying Multidimensional LiquidChromatographic Proteomics WithoutIsotopesHua Lin, Weixun Wang, Sushmita Roy, Thomas A. Shaler,Haihong Zhou, Lander R. Hill, Markus Anderle,Praveen Kumar, and Christopher H. Becker

SurroMed, Inc., Mountain View, CA

Differential protein quantification by mass spectrometry has received sub-stantial attention for gel-less (image-less) separations. Approaches oftenhave involved tagging of specific amino acids with isotopically labeledreagents, or use of isotopically labeled or chemically modified standardreference materials. In contrast, our approach does not use additionalchemicals or manipulations. It is based on making the analytical processhighly reproducible and the fact that signal response is generally linear asa function of concentration even for highly complex matrices. (This methodis also applicable to metabolomics.)

Various validation studies for the method will be shown along with aclinical study of rheumatoid arthritis (RA) involving 12 human subjects.While the choice of chromatographic methods may vary, detailed resultsfrom the RA study using a combination of strong-cation-exchange (off-line) and reverse-phase (on-line) show the tracking and quantification of34,000 de-isotoped molecular ions per sample with human serum con-sumption of 30 microliters. Median coefficient of variation was 26%.Differential expression measurements on these RA samples vs. healthycontrols confirm of previously reported putative biomarkers and revealnew unanticipated markers.

A.6Strategies to Monitor Structurally AlteredEstrogen Receptor as Found in BreastCancersChristian Atsriku1, Jose E. Meza2, Gary K. Scott1,Christopher C. Benz1,3, and Michael A. Baldwin4

1Buck Institute, Novato, CA; 2Agilent Technology, Santa Clara, CA;3Comprehensive Cancer Center & Division of Onco-Hematology,University of California, San Francisco, CA; 4Mass SpectrometryFacility, Department of Pharmaceutical Chemistry, University ofCalifornia, San Francisco, CA

Oxidant-induced structural modifications within the cysteine-rich DNA-binding domain (DBD) of the overexpressed estrogen receptor (ER) likelycontribute to its loss of DNA-binding function and altered transcriptionalactivity during human breast cancer development. Thiol specific oxidationof ER may account at least partially for the fact that the otherwise intact(67kDa) and immunoreactive ER present in about one-third of all ERoverexpressing primary breast tumors, appears to be completely unableto bind DNA [1]. This DNA-binding defect has been demonstrated in vitroby thiol specific oxidation or alkylation of ER-enriched breast cancerextracts or purified recombinant ER protein [2]. By using recombinant(67kDa) ER as a model, we have developed and validated protocols todetect endogenously produced oxidative modifications of cysteine resi-dues in the two Cys4-type zinc fingers within the DBD of ER extractedfrom breast cancer cells. Unfortunately, routine ER extraction and purifi-cation results in the ex vivo oxidation of these ER-DBD cysteine residues.These problems can be circumvented by an initial irreversible alkylation ofall free thiols followed by reduction of any disulfides and treatment withchemically identical but isotopically different alkylating agents. The oxida-tion state of recombinant ER was probed by differential alkylation, beforeand after reduction followed by a two-enzyme digestion using endopro-teinase Lys-C and Asp-N. The labelled peptides were subsequently ana-lyzed and quantitated by HPLC/ESI-MS. Alkylation with [13C2]-bromoace-tic acid (BAA**) and non-isotopically labelled iodoacetic acid (IAA) resultedin chemically identical but isotopically different carboxymethylation of freecysteines. Digestion of ER with the above enzymes produced four diag-nostic peptides, each of which contained two cysteine residues and gavevariable ESI-MS detection sensitivities. Incomplete alkylation of cysteineresidues within one of the two zinc fingers necessitated the use of adenaturant (guanidine HCl), during the initial alkylation process while thesecond alkylation (involving the SDS-PAGE purification protocol) wasperformed during the enzyme digestion step. To validate the method,artificial oxidation was induced by titrating ER with a cysteine-specificoxidant (diamide) as a function of concentration and time. The relativeamounts of each oxidized species were determined from mass spectro-metric peak heights. These analytical strategies will be employed to mon-itor structurally altered estrogen receptor produced within oxidantstressed human breast cancer cells.

Supported by NIH CA71468, DOD DAMD17-99-1-9111, and NCRRRR01614.

1. Baldwin, M. A., and Benz, C. C. (2002) Meth. Enzymol. 358, 54–69.2. Liang, X., Lu, B., et al. (1998) Mol. Cell. Endocrinol. 146, 151–161.




http://ww

w.m

cponline.org/D

ownloaded from


A.7Development of a Fully AutomatedTwo-dimensional Nano-electrosprayLC/MS System for Sub-fmol ProteomicsAnalysisTerry Zhang, Ken Miller, and Dirk Chelius

ThermoFinnigan, San Jose, CA

Introduction: Two-dimensional LC/MS methods for proteomic analysis ofcomplex peptide mixtures from protein digests are starting to replacetypical two-dimensional gel electrophoresis. Nano-electropray coupledwith mass spectrometry is the method of choice for maximum sensitivity.The highest sensitivity is important for the detection of low abundanceproteins.

Methods: In this study we describe a fully automated two-dimensionalnano-electrospray LC/MS system. A strong cation exchange (SCX) col-umn is used as the first dimension and the peptides are eluted onto apeptide-trap at a high flow rate. A second pump is used at low flow rate(200 nl/min) to elute the peptides from the peptide trap and for theseparation of the peptides on a reversed-phase column (75 �m ID) as asecond dimension. The PicoFritTM column is placed directly in front of themass spectrometer, limiting the peak dispersion after the column. Whilepeptides are eluted from one peptide trap and PicoFritTM column, asecond peptide trap is loaded with peptides from the SCX column, reduc-ing the analysis time in half.

Results: Compared to the two-dimensional micro-spray system, thenanospray system showed increased peptide coverage and increasedprotein identification in complex mixtures. Peptides from a bovine serumalbumin digest could be detected at levels as low as 500 attomol, dem-onstrating the high sensitivity of the described method. This methodcombines fast separation of peptides in a first dimension at high flow rateand high sensitivity detection of peptide fragments using the PicoFrit™column at low flow rate in a second dimension.

A.8Mass Spectrometry Based Scanning forPost-translational Modifications ofHistonesPatrick Schindler, Harry Towbin, Michele Coulot,Otto Zingel, and Jan van Oostrum

Novartis Pharma AG, Basel, Switzerland

The earlier view of chromatin as a mere structural entity has changed inrecent years towards the idea that histones have a much more dynamicrole in controlling gene activity. DNA is wrapped around a set of so-calledcore histones, H2A, H2B, H3 and H4, to form an octamer. This stretch ofDNA consists of 146 base pairs which together with the histones form thebasic unit of chromatin, the nucleosome. An additional histone, H1, bindsto these nucleosomes, leading to a further level of organization.

Histones are known to be subject to a large variety of post-translationalmodifications that have with time been recognized as being crucial foractivation of transcription as well as silencing of genes. The modificationsdescribed affect lysines (acetylation, mono-, di-, and trimethylation, ubiq-uitination), serine and threonine (phosphorylation), and arginines (mono-and two types of dimethylation) (1). Most of these modifications are readilyreversible at the level of the nucleosome. The enzymatic machinery re-sponsible for the modifications as well as their reversal is very complexand only partly understood. This also holds for the signaling mechanismscontrolling these enzyme complexes. One concept currently evolving isthat the histone modifications form a code that allows binding of particularproteins involved in further modification of the histone tail and finally,transcription or silencing of a gene.

In particular, acetylation of the N-terminal tails of histones H3 and H4,which remain accessible in the nucleosome, have been extensively stud-ied (2). Acetylation leads to a loosening of the nucleosome and is associ-ated with actively transcribed genes. In contrast, deacetylation is con-nected with repression and silencing. Integration of all these phenomenatowards a coherent biological mechanism remains a major challenge.

One necessary step towards this goal consists of a comprehensiveanalysis of all the histone modifications in their biological context. MALDI-TOF and nano-ESI mass spectrometry are especially suited to the analysisof post-translational modifications (3), e. g., phosphorylation of serine,threonine and tyrosine residues. These have been shown to give rise to adiagnostic fragment ion (m/z 79) in the negative mode that enables spe-cific detection of phosphopeptides in a mixture (4). Recently mass spec-trometry-based techniques based on diagnostic fragment ions have alsobeen used to characterize the methylation and acetylation content of corehistones (5, 6).

We will report here on the use of these mass spectrometric techniquesin combination with affinity purifications, enzymatic digestions, and HPLCseparations for the identification of post-translationally modified sites incore histones.

1. Fischle, et al. (2003) Curr. Opin. Cell Biol. 15, 172–183.2. Lachner, M., and Jenuwein, T. (2002) Curr. Opin. Cell Biol. 14,

286–298.3. Schweppe, et al. (2003) Accounts Chem. Res., in press.4. Annan, et al. (2001) Anal. Chem. 73, 393–404.5. Zhang, et al. (2002) Anal. Biochem. 306, 259–269.6. Kim, et al. (2002) Anal. Chem. 74, 5443–5549.




http://ww

w.m

cponline.org/D

ownloaded from


A.9Effects of Different Ventilation Strategieson the Proteome of Rat Alveolar Type IICellsJan Hirsch1, Kirk C. Hansen2, James A. Frank1,Robert J. Chalkley2, Xiaohui Fang1, Alma L. Burlingame2,and Michael A. Matthay1

1Cardiovascular Research Institute, University of California, SanFrancisco, CA; 2Mass Spectrometry Facility, Department ofPharmaceutical Chemistry, University of California, San Francisco,CA

To identify changes in the proteome of alveolar type II cells (ATII) afterventilation with high and low tidal volume, rats were mechanically venti-lated for 5 h with a high tidal volume (20 ml/kg without PEEP) or a low tidalvolume (6 ml/kg with PEEP 4 cm H2O) and compared to controls withoutmechanical ventilation. ATII were isolated (purity 90%), lysed by sonicationand fractionated. After labeling using cleavable isotope coded affinity tags(ICAT), tryptic digestion and cation exchange chromatography, the biotin-tagged cysteine containing peptides were extracted using an avidin-coated column and identified by electrospray MS (ESI-QTOF) and proteinsequencing by collision induced dissociation. Spectra were interrogatedagainst the NCBI database and quantified using LC-BatchTag and MS-ICAT from the ProteinProspector suite of programs. Quantitation showeddifferential expression of a large number of proteins following both highand low tidal volume ventilation, including up-regulation of ICAM-1, ribo-somal proteins and Na�, K�-ATPase. The upregulation of beta-defensin,lysozyme, ribosomal proteins and Na�, K�-ATPase was markedly reducedafter high tidal volume compared to low tidal volume ventilation. Weconclude that differences in protein expression in ATII induced by me-chanical ventilation can be detected by ICAT and MS. The identifiedproteins are important in the innate immunity system and in the clearanceof pulmonary edema fluid. The attenuated response of ATII after ventilationwith high tidal volume may indicate a clinically relevant impediment of hostdefense against pathogens. Supported by NIH HL 69900, NIH HL51854,NCRR 01614, DFG HI 810/1, and the Vincent J. Coates Foundation.

A.10Enhanced Throughput and Sensitivity inMALDI Mass SpectrometryXiaoxi (Kevin) Chen, Neia Illingworth, Michael Shanler, andKevin M. Hughes

BD Biosciences Discovery Labware, Bedford, MA

Sample preparation for MALDI mass spectrometry usually includes puri-fication and concentration of protein/peptide samples. This is followed bymixing the samples with MALDI matrices and spotting on targets. Exten-sive and time-consuming preparation work is required to obtain optimalsensitivity. As proteomics research intensifies, researchers hope to ana-lyze more samples daily and detect less abundant proteins/peptides. Anovel method utilizing an elastomeric multi-channel device (patent pend-ing) has been developed at BD Biosciences Discovery Labware to in-crease throughput, enhance sensitivity, and improve reproducibility forMALDI sample preparation.




http://ww

w.m

cponline.org/D

ownloaded from


A.11Taggor-PepMap, a New Software PipelineUsing LC-MS/MS Data for GenomeAnnotationErwan Reguer1, Myriam Ferro2, Estelle Nugues1,Thierry Vermat3, Romain Cahuzac2, Marielle Vigouroux3,Emmanuelle Mouton2, Yves Vandenbrouck3, Jerome Garin2,and Alain Viari1

1INRIA, Montbonnot, France; 2Laboratoire Chimie des Proteines,CEA/INSERM- CEA/Grenoble, Grenoble, France; 3GENOME Express,Meylan, France

Many highthroughput proteomic strategies involve LC-MS/MS analyses oftrypsin digests derived from complex protein mixtures. Subsequent data-base searching uses search engines, such as Mascot or Sequest, whichcarry out identity searching without de novo sequencing. In most cases,protein databases to be searched are an annotated version of the corre-sponding genome. Indeed many of the proteins present in these data-bases derive from software predictions and are referred to as hypotheticalor putative proteins. But many annotation errors exist. For instance it wasestimated that at least 30% of the A. thaliana genome is not properlyannotated. One of the present challenges of proteomics is to identify minorproteins as well as those that are difficult to analyze, such as membraneproteins. Because, generally, few biochemical studies are performed onthese proteins, these are liable to be incorrectly predicted. Thus in order tobe able to identify new proteins, from highthroughput proteomic studies,we developed a software pipeline, called Taggor-PepMap. This pipelineuses MS/MS data to mine not only protein databases but also genomicinformation. Thus Taggor-PepMap is an integrated tool that allows a directcorrelation between expressed proteins and genomic information. In thatrespect, Taggor-PepMap provides valuable information for gene localiza-tion and genome annotation. This pipeline software is made of two mod-ules. A list of peptide sequence tags (PSTs) is generated from MS/MS dataand is used to mine protein and genomic databases. A PST is defined asa three amino acid peptide sequence and by two masses correspondingto the two adjacent stretches af amino acids (Mann, 1994). The firstmodule, Taggor, allows PSTs to be generated automatically from LC-MS/MS data obtained on a QTOF type instrument. Then PSTs are eithermapped on protein sequences or on the six translated phases of agenomic sequence, taking into account complete or partial PSTs. Finally aclustering phase allows PSTs to become affiliated to a same protein orgene. The mapping and the clustering phases are implemented in thePepMap software. PepMap was independently assessed using virtual andgenuine PSTs retrieved from A. thaliana. Using the same experimentaldata, Taggor was also evaluated for its ability to generate correct PSTs.The complete pipeline was eventually tested and proved to be fast andreliable. The Taggor-PepMap pipeline was used in the context of the studyof the A. thaliana chloroplast envelope (Ferro, accepted) and demon-strated its usefulness for genome annotation.

A.12Proteomic Analysis of Human Serum by2D DIGE and SELDI-MSArlene D. Gonzales, Brett A. Chromy, Gloria A. Murphy,Megan W. Choi, and Sandra L. McCutchen-Maloney

Lawrence Livermore National Laboratory, Livermore, CA

We have applied two proteomic technologies, 2D DIGE (Differential In GelElectrophoresis) and SELDI (Surface Enhance Laser/Desorption Ioniza-tion) mass spectrometry to characterize human serum. Here, we presentmethods used. Because albumin is a major protein constituent of serum,we tested various removal methods and compared albumin removal effi-cacy and overall protein yield/sample loss. Serum samples with andwithout albumin removal were analyzed by two-dimensional electrophore-sis using novel DIGE technology (Amersham), which allows multiplexanalysis of up to three different serum samples on one gel, therebyreducing gel-to-gel variability and increasing statistical significance ofresults. These methods were applied to HUPO (Human Proteome Orga-nization) reference serum samples, in addition to “diseased” serum sam-ples of interest in our laboratory. Our “diseased” samples were comparedto “normal/control” serum with the overall goal of identifying biomarkersassociated with disease. Differentially expressed serum proteins wereidentified by MALDI-MS and/or tandem MS (Proteomic Research Servic-es). In parallel, SELDI mass spectrometry was also used to monitor dif-ferent protein expression patterns in serum. Pre-fractionation of serumsamples using an anion exchange column was followed by SELDI-MSanalysis using an IMAC copper affinity chip. Our results demonstrate that2D DIGE and SELDI-MS provide a powerful combination for proteomicanalyses for human serum, and this approach promises to identify diverseprotein biomarkers in human serum for a variety of diseases andconditions.

This work was performed under the auspices of the U.S. Department ofEnergy by University of California Lawrence Livermore National Laboratoryunder contract No. W-7405-ENG-48 with support from the Department ofHomeland Security (Biological Warning and Incident Characterization Pro-gram). UCRL-JC-153605-ABS.




http://ww

w.m

cponline.org/D

ownloaded from


A.13Subcellular Proteomic Characterization ofHost Response to Yersinia pestis andNear NeighborsCelia G. Zhang, Arlene D. Gonzales, Megan W. Choi, andSandra L. McCutchen-Maloney

Lawrence Livermore National Laboratory, Livermore, CA

Yersinia pestis is the causative agent of plague and a known biothreatagent. Y. enterocolitica and Y. pseudotuberculosis are also human patho-gens in the genus Yersinia. A better understanding of the differences inhost response to these closely related human pathogens can 1) bettercharacterize the different mechanisms of virulence and pathogenesis, 2)provide biomarkers for early detection of exposure to biothreat agents,and 3) define novel therapeutic targets for treatment of plague. Resultspresented here focus on subcellular proteomic characterization of hostresponse to these three closely related human pathogens using a humanmonocyte cell-line (U937).

Specifically, protein fractionation methods were used to monitor hostprotein expression changes, post pathogen exposure, at the cytoplasmic,nuclear and membrane subcellular levels. Methods were optimized toprevent cross contamination of cytoplasmic and nuclear fractions. In orderto achieve a well-focused first-dimension separation, membrane proteinswere resolubilized using an optimized buffer that maintains solubilizationof disaggregated proteins throughout electrophoresis. 2-dimensionalDIGE (differential in gel electrophoresis) using three different fluorescentdyes was used to identify statistically significant up-regulated or down-regulated host proteins in the subcellular fractions after pathogen expo-sure across a range of pathogen exposure conditions. The use of pooledinternal standard dramatically reduced experimental variations acrossmultiple experiments. Changes in protein levels were detected and quan-titated using the DeCyderTM software platform. Differentially expressedproteins were identified by MALDI-MS and/or tandem MS (ProteomicResearch Services). By characterizing subcellular fractions, it is possibleto assign specific cellular localization of proteins of interest and to monitorprotein trafficking/signaling within a cell after pathogen exposure, thusproviding a better mechanistic understanding of pathogenesis for Y. pestisand near neighbors.

This work was performed under the auspices of the U.S. Department ofEnergy by University of California Lawrence Livermore National Laboratoryunder contract No. W-7405-ENG-48 with support from the Department ofHomeland Security (Biological Warning and Incident Characterization Pro-gram) UCRL-JC-153607-ABS.

A.14Modification of Bovine Cytochrome cOxidase by Peroxide and 4-Hydroxy-2-Nonenal: Changes in Protein andCardiolipin StructureSusan T. Weintraub, Andrej Musatov, Christopher A. Carroll,and Neal C. Robinson

Department of Biochemistry, The University of Texas HealthScience Center, San Antonio, TX

Cytochrome c oxidase (CcO), the terminal enzyme in the electron trans-port chain in eukaryotes and many bacteria, catalyzes a four electronreduction of oxygen to water with the simultaneous translocation of pro-tons across the membrane, generating a proton gradient that drives ATPsynthesis. CcO consists of 13 dissimilar subunits and at least four mole-cules of bound cardiolipin (CL), with a combined molecular weight of200,000 Da. We have been investigating structural alterations that occuras a consequence of direct exposure of CcO to peroxide and lipid per-oxidation products. We have previously reported that after incubation with4-hydroxy-2-nonenal (HNE), CcO exhibits a dose-dependent loss of bio-logical activity, with covalent attachment of HNE occurring on predomi-nantly three subunits: VIIa, VIIc, and VIII. Through a combination of MALDI-TOF/MS and ESI/MS3 we were able to identify His-36 as the specific siteof modification of subunit VIII, the subunit which became modified to thegreatest extent under our experimental conditions. Hydrogen peroxide(H2O2) is a strong cellular oxidant that is a major causative agent ofoxidative damage to proteins and phospholipids. It has been shown thatthe binuclear site of bovine CcO reacts with hydrogen peroxide leading tothe generation of two intermediates with absorption maxima at 580 nmand 607 nm. We have recently determined that a number of irreversiblestructural and functional changes occur when CcO is treated with H2O2 ortert-butyl hydroperoxide (t-BOOH). The electron-transport activity of CcOwas found to decrease by as much as 80% with H2O2 and 40% witht-BOOH, both in a time- and concentration-dependent manner. Structuralmodification of CcO by peroxide was initially detected by reversed-phaseHPLC. MALDI-TOF/MS and ESI/MS analysis of peroxide-treated CcOrevealed that two subunits, VIIc and IV, became modified (addition of anoxygen atom to each) after treatment with either H2O2 or t-BOOH. Throughuse of ESI/MS/MS, it was found that the sites of oxidation were Trp-19 onsubunit VIIc and Trp-48 on subunit IV. The extent of subunit VIIc modifi-cation correlated well with percent CcO inactivation. In addition to perox-ide-induced alterations of these subunits, modification of the CL bound toCcO was also observed in a concentration-dependent manner that cor-related with the decreases in CcO electron-transport activity. We concludethat the inhibition of CcO by peroxides occurs by two mechanisms:modification of subunit VIIc and/or by peroxidation of tightly bound CL.

This work was supported by the American Heart Association-TexasAffiliate (0160115Y), National Institutes of Health (GMS 24795) and RobertA. Welch Foundation (AQ1481).




http://ww

w.m

cponline.org/D

ownloaded from


A.15Proteomic Analysis of the CompleteRibosome from Rhodopseudomonaspalustris Using Integrated “Top-down”and “Bottom-up” Mass SpectrometricApproachesMichael Brad Strader1, Nathan C. VerBerkmoes1,Robert L. Hettich1, John W. Barton2, Barry D. Bruce3,Loren J. Hauser2, Frank W. Larimer2, Brian H. Davison2,Dale A. Pelletier2, and Gregory B. Hurst1

1Chemical Sciences Division; 2Life Sciences Division; Oak RidgeNational Laboratory, Oak Ridge, TN; 3Department of Biochemistryand Cellular and Molecular Biology, University of Tennessee,Knoxville, TN

To better understand the nature of how the ribosome functions in trans-lating the mRNA template into functional proteins, a detailed knowledge ofall the protein components involved in this process is imperative. To thisend, we present a proteomic study involving a comprehensive massspectrometric approach that integrates intact protein molecular massmeasurement (top-down) and proteolytic fragment characterization (bot-tom-up) of the complete ribosome from the prokaryote Rhodopseudomo-nas palustris. Because R. palustris is a facultative photoheterotroph weused this integrated approach to study ribosomes purified from cellsgrown under both aerobic and anaerobic conditions. In this study, we haveidentified all 54 E. coli orthologs to ribosomal proteins. Furthermore, allidentifications, with the exception of L32 and L36, were based on tandemmass spectrometric identification of 2 or more unique peptides per ribo-somal protein, indicating a reasonable level of certainty for each identifi-cation. Interestingly, proteomic analysis identified 5 novel proteins thatconsistently co-sediment in sucrose gradient centrifugation with ribo-somes from anaerobically grown R. palustris that are not found underaerobic growth conditions. To further characterize ribosomal proteins weare currently using data from both top-down and bottom-up analyses toconfidently identify posttranslational modifications. To our knowledge, thisstudy represents the only comprehensive analysis to date that integratesboth approaches for studying a prokaryotic ribosome.

A.16Enzymatic and Non-enzymatic Acetylationof the HIV-1 Tat Protein: An In Vitro StudyWilma Dormeyer1, Alexander Dorr2, Melanie Ott2,and Martina Schnoelzer1

1Protein Analysis Facility; 2Applied Tumor Virology; DKFZ (GermanCancer Research Center), Heidelberg, Germany

The human immunodeficiency virus-1 transactivator of transcription(HIV-1 Tat) is a small nuclear protein that stimulates transcriptional elon-gation by transiently binding to an RNA stem loop structure called thetransactivation-responsive element (TAR) which is located at the 5� end ofall viral transcripts. The first 72 amino acids of Tat are sufficient to trans-activate transcription from the viral long-terminal repeat (LTR) and com-prise five regions: an N-terminal acidic region, a cysteine-rich region(CRR), a core region, an arginine-rich motif (ARM) and a glutamine-richregion. The CRR and the core region form the cofactor binding regionwhich interacts with cofactors such as cyclin T1 whereas the ARM isessential for TAR binding. Here, we report on the acetylation of Tat by theacetyltransferase activity of p300.

In vitro acetylation assays using synthetic peptides corresponding todifferent Tat regions were performed in the presence of p300 acetyltrans-ferase enzyme and acetyl-coenzyme A (AcCoA) or with AcCoA alone asnegative control. Analysis of the acetylation reactions by MALDI TOF MSrevealed that the ARM is the only acteylation target of p300 in Tat.Proteolytic digestion in combination with MALDI-TOF MS and sequentialEdman degradation mapped the acetylation site of p300 to a single lysineresidue, K50, in the Tat ARM.

Surprisingly, we also observed a strong acetylation reaction in theabsence of p300 mapping to the Tat CRR and to a lesser extent of the Tatcore region. MALDI TOF MS of the acetylated Tat CRR indicated that atleast three residues were acetylated, exceeding the number of lysines(28K29K) in this region. Furthermore, multiple CRR acetylation occurredindependently from the presence of lysine residues since a mutant peptidein which 28K29K were mutated to alanines showed the identical acetyla-tion pattern as the wild type CRR peptide. However, chemical protectionof all cysteine residues in the CRR peptide completely inhibited the acety-lation reaction implying that an enzyme-independent transfer of the -SHcoupled acetyl group from Acetyl-CoA to the side chain of the Tat cysteineresidues was responsible for the Tat CRR acetylation.

In terms of biological relevance, a coenzyme function of Tat in acetyl-transferase reactions might be considered and will be tested in futuretransacetylation experiments. In general, being aware of the cysteinecontent of peptides and proteins is important for the interpretation of invitro acetylation assays and the mapping of acetylation sites.




http://ww

w.m

cponline.org/D

ownloaded from


A.17Mapping O-GlcNAc Modification Siteswith �-elimination/Michael Addition UsingDTT as an Affinity TagKeith Vosseller1, Lance Wells2, Bob Cole2, Kirk Hansen1,Robert Chalkley1, Janet Cronshaw2, Michael J. Matunis2,A. L. Burlingame1, and Gerald Hart2

1Mass Spectrometry Facility, University of California, San Francisco,CA; 2Johns Hopkins University, Baltimore, MD

Identifying sites of post-translational modifications on proteins is a majorchallenge in proteomics. O-GlcNAc is a dynamic nucleocytoplasmic mod-ification more analogous to phosphorylation than to classical complexO-glycosylation. We describe a mass spectrometry based method for theidentification of sites modified by O-GlcNAc that relies on mild �-elimina-tion followed by Michael addition with dithiothreitol (BEMAD). The modi-fied peptides can be efficiently enriched by affinity chromatography. WhileO-GlcNAc is quite labile in mass spectrometry, the DTT replacement isrelatively stable during CID, allowing for more information about specificsites of modification. This same methodology can be applied to mappingsites of serine and threonine phosphorylation and we provide a strategythat uses modification specific antibodies and enzymes to discriminatebetween the two post-translational modifications. The BEMAD methodwas validated by mapping three previously identified O-GlcNAc sites, aswell as three novel sites, on Synapsin I purified from rat brain. BEMAD wasthen used on a purified nuclear pore complex (NPC) preparation to mapnovel sites of O-GlcNAc modification on the lamin B receptor and thenucleoporin Nup155. The method is shown to be amenable to performingquantitative mass spectrometry with the use of differential isotopic ver-sions of DTT. In addition, our studies emphasize the importance of dis-tinguishing between O-phosphate versus O-GlcNAc when mapping sitesof serine and threonine post-translational modification using �-elimina-tion/Michael addition methods.

Supported by NCRR RR01614.

A.18Mass Spectrometry Characterization ofHuman Erythrocyte SpectrinPhosphorylation Sites and Side-reactionsof NTCB CleavageHsin-Yao Tang and David W. Speicher

The Wistar Institute, Philadelphia, PA

Spectrin is the major component of the erythrocyte membrane skeletonand exists as a 526 kDa �� heterodimer. The 246 kDa �-chain is phos-phorylated near the C-terminus but the number of phosphorylation sitesand their specific locations has not previously been identified. In thisstudy, we isolated the phosphorylated region of �-spectrin using 2-nitro-5-thiocyanobenzoic acid (NTCB), which cleaves the peptide bond on theN-terminal side of cysteine residue, followed by immunoaffinity purifica-tion. Initial NTCB cleavage reactions using established protocols resultedin a number of side-reactions that confounded the analysis of spectrinphosphorylation patterns on two-dimensional gels. Analysis of the NTCBcleavage products from either a recombinant protein or a synthetic modelpeptide by matrix-assisted laser desorption/ionization mass spectrometry(MALDI MS), liquid-chromatography tandem MS (LC-MS/MS) and re-versed-phase high performance liquid chromatography (RP-HPLC) iden-tified carbamylation of lysine and cyclization of cyanylated cysteine as themajor NTCB side-reactions. The NTCB reaction was subsequently opti-mized to generate cleavage products with high yield and homogeneity.Two-dimensional gel analysis of the immunopurified NTCB products oferythrocyte spectrin revealed nine charge states, suggesting the presenceof up to eight phosphorylation sites. Most of the proteins had one to fourphosphates per molecule and only a small fraction was unphosphorylatedor had more than four phosphates. The phosphorylated residues wereidentified using a combination of immobilized metal affinity chromatogra-phy (IMAC), MALDI MS, and LC-MS/MS. Quantitative analysis of thephosphorylation states by RP-HPLC tryptic peptide mapping revealed thatphosphorylation of �-spectrin occurred in a sequential manner where eachspecific site was completely phosphorylated before the next site wasmodified. The first phosphorylation event occurs on Ser-2114, followed bySer-2125, Ser-2123, Ser-2128, Ser-2117 and Thr-2110. These six phos-phorylation events account for at least 96% of the spectrin molecules infreshly isolated human red cells. Although the charge states observed ontwo-dimensional gels suggest that up to two additional minor phospho-rylated sites may occur, it is more likely that these additional spots arecaused by minor non-phosphate modifications of this region of theprotein.




http://ww

w.m

cponline.org/D

ownloaded from


A.19Whole Blood as a Human Model forProteomic Host Response to PathogensSandra L. McCutchen-Maloney, Gloria A. Murphy,Arlene D. Gonzales, Jenny L. Heidbrink, Megan W. Choi,and Brett A. Chromy

Biodefense Division, Lawrence Livermore National Laboratory,Livermore, CA

Models for human disease typically rely on the use of cell culture or animalmodels. Here, we report the use of human whole blood as a means tostudy host-pathogen interactions. Specifically, whole blood was used tocharacterize proteomic host response to infectious agents including Y.pestis, the causative agent of plague, and near neighbors Y. enterocoliticaand Y. pseudotuberculosis, also human pathogens that unlike Y. pestis,are rarely fatal. Classical proteomic 2-dimensional electrophoresis hasproven largely unsuccessful for detecting differential protein expression inblood due to several factors including high abundance proteins such asalbumin and hemoglobin that result in crowding of 2DE separated proteinspots. To address these issues, we have analyzed fractionated whiteblood cells and serum, post albumin removal. Differential expression wasmonitored by 2D DIGE (Differential In Gel Electrophoresis) and SELDI(surface enhanced laser/desorption ionization) mass spectrometry in orderto detect proteomic changes following pathogen exposure. Proteins wereidentified by MALDI-MS and/or tandem MS (Proteomic Research Servic-es). Examination of the differentially expressed proteins after exposure tothe three pathogens reveals shared expression changes indicative of acommon immune response; however, distinct pathogen-specific expres-sion differences were also observed. These pathogen-specific proteomicchanges distinguish the three different pathogen exposures and representnovel detection biomarkers for exposure to plague. Additionally, thecomparative characterization of proteomic host response provides in-sight into the different virulence and pathogenic mechanisms underlyingthese three genetically similar pathogens with significantly differentclinical manifestations.

This work was performed under the auspices of the U.S. Department ofEnergy by University of California Lawrence Livermore National Laboratoryunder contract No. W-7405-ENG-48 with support from the Department ofHomeland Security (Biological Warning and Incident Characterization Pro-gram). UCRL-JC-153606-ABS.

A.20Pneumatic Assistance in MALDI—TheProperties of Commonly-used CarboxylicAcid MatricesVictor L. Talroze1, A. L. Burlingame1, Ilya Leipunsky2, andMichael A. Baldwin1

1Department of Pharmaceutical Chemistry, University of California,San Francisco, CA; 2Russian Academy of Sciences, Moscow,Russia

We have developed a self-consistent quantitative theory of MALDI as athermal process in which CO2 is released by laser irradiation of carboxylicacid matrices, resulting in pneumatic assistance (Talroze et al., Adv. MassSpectrom. 2000). Mass and heat transfer equations model the matrixcomposition, the degree of melting and pyrolysis, and the amount of gasin the melt at any time and at any depth during or after the typical 3 nspulse from a nitrogen laser at 337 nm. Gas diffusion results in bubbleformation, growth, eventual bursting and sputtering of molten matrix en-training analyte ions formed during crystallization. The model relies onmacroscopic properties of the matrix and its decarboxylation product, andcalculates and predicts a temporal and spatial model of the following:

• Absorption of laser radiation by the matrix, with some loss of energyby fluorescence.

• The effects of heating, melting, decarboxylation and subsequentcooling after the laser pulse.

• Super-saturation of molten matrix and the decarboxylation productwith CO2.

• Genesis, growth and bursting of bubbles in the melt, and the flight offragments as a plume of droplets.

• Competing loss of analyte ions by recombination of ion pairs andsingle ions, both in the body of the melt and in the flying microdro-plets of the plume.

• Partial or total evaporation of the matrix and its decarboxylationproduct from the analyte ions.

It is of interest to determine whether this theory can explain differences inthe MALDI characteristics of known matrices such as �–cyano-4-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). DHB is arelatively “soft” matrix compared with CHCA, i. e., it induces less frag-mentation in the analyte ions, even though the translational energy of DHBclusters in the MALDI plume is higher than that of CHCA. Although DHBhas the lower melting point by �45 °C, its substantially lower molarabsorption coefficient at 337 nm necessitates the use of higher laserfluence in order to achieve melting and dissociation. Thus the depth of themolten layer is greater for DHB than for CHCA, although the melt may becooler. There are significant gaps in the known literature values for thephysical and thermochemical properties of these compounds and theirdecarboxylation products, but we will attempt to use the thermal pneu-matic assistance model to compare the behavior of DHB and CHCA asMALDI matrices. Such considerations may play an important role in thefurther optimization of this important analytical method.

We acknowledge support from NIH RR01614.




http://ww

w.m

cponline.org/D

ownloaded from


A.21Affinity Capture of Ligands fromBrain-conditioned Medium by NuclearReceptors: Polyunsaturated Fatty AcidsBind to RXR AlphaJohan Lengqvist1, Jan Sjovall1, Thomas Perlmann2,and William J. Griffiths3

1Karolinska Institutet, Stockholm, Sweden; 2Ludwig Institute forCancer Research, Stockholm, Sweden; 3School of Pharmacy,University of London, London, UK

A number of reports have shown that expressed nuclear receptor (NR)ligand binding domain (LBD) proteins can be purified with lipids boundwithin the ligand binding pocket (Bourguet W, et al. 2000. Mol Cell 5, 289:Wisely GB, et al. 2002. Structure 10, 1225). In the current work, an affinitycapture method based on the assumption that lipids selectively bound inthe hydrophobic ligand binding pocket of a NR-LBD may survive proteinpurification has been developed. The retinoid X receptor (RXR) alpha LBDwas used as an affinity handle to capture ligands from the brain-condi-tioned medium. After incubation of the RXR-LBD with brain-conditionedmedium for 30 min at 37 °C, the protein was purified on a Ni-NTA agarosecolumn and then desalted on a PD-10 gel filtration column. Lipids boundto the purified protein were released by denaturing the protein, and weresubsequently extracted using a Lipidex-1000 gel. The extracted lipidswere analysed by nano-ES-MS and MS/MS on a Quattro Micro triple-quadrupole instrument. Three major [M-H]- ions were observed, corre-sponding to deprotonated molecules of oleic, arachidonic and decosa-hexaenoic acids. In contrast, when the RXRa was purified after incubationwith non-conditioned medium, these [M-H]- ions were not observed (orwere in very low abundance). This result suggests that polyunsaturatedfatty acids are released from brain complex-lipids during incubation in themedium, and that their binding to the RXRa LBD is sufficiently strong towithstand the protein purification procedure. To show binding specificityof the fatty acids, a 1.5-fold excess of high affinity ligand LG268 wasadded to the medium prior to the addition of RXR-LBD. Formation of theRXRa LBD - fatty acid complex was inhibited, as would be expected if thefatty acids were acting as true ligands competing with LG268 for bindingto the RXRa ligand-binding pocket.

The results of the present study were further corroborated by activationstudies on transfected cells (Mata de Urquiza A et al. (2000) Science 290,2140) and electrospray studies of RXRa LBD - fatty acid complexes.

In conclusion, we have described an affinity capture method for theidentification of ligands to RXR. We are currently extending this work toinvestigate ligand binding to other nuclear receptor proteins.

A.22Analysis of Multi-protein Complexes andWhole Cell OrganellesJoerg Reinders1, Yvonne Wagner1, Rene Zahedi1, CorneliaJoppich2, Helmut E. Meyer2, Peter Rehling3, Nikolaus Pfanner3,Chris Meisinger3, and Albert Sickmann1

1Rudolf-Virchow-Center for Experimental Biomedicine, UniversitaetWuerzburg, Wuerzburg, Germany; 2Medizinisches Proteom-Center,Ruhr-Universitaet Bochum, Universitaetsstrasse 150, Bochum,Germany; 3Institut fuer Biochemie und Molekularbiologie,Universitaet Freiburg, Freiburg, Germany

The analysis of complex protein mixtures like ribosomes, centrosomes oreven whole cell compartments is still a difficult problem. In general suchanalyses employ a high resolution technique, e.g. 2D Polyacrylamide GelElectrophoresis (2D-PAGE), followed by protein identification via massspectrometry (peptide mass fingerprinting). The 2D-PAGE is a time con-suming method for separation and is not suitable for all kinds of proteins,e. g., hydrophobic proteins, basic proteins, very large or very small pro-teins. Another possibility for analysis of highly complex protein mixtures ismultidimensional LC-ESI-MS/MS (peptide fragmentation fingerprinting).Data collection and interpretation has to be automated due to the enor-mous numbers of MS/MS-spectra. To study these problems we combinedseveral separation methods like 1D and 2D PAGE combined with peptidemass fingerprinting using MALDI-MS as well as 1D and 2D chromatogra-phy coupled online to an ESI-ion trap mass spectrometer collecting thespectra automatically. For 2D-HPLC an orthogonal system was usedconsisting of a capillary ion exchange in the first and a nano-reversedphase chromatography in the second dimension. After the first dimensionthe sample was cleaned up in a preconcentration step. The methods werevalidated using a tryptic digest of yeast ribosomes and than applied toyeast mitochondria. A combination of all methods showed very goodcoverage for most proteins.

A.23MALDI-TOF-MS Differentiation of YeastsRichard O. Jenkins1 and Richard E. Sherburn2

School of Molecular Sciences, De Montfort University, Leicester,UK; 2School of Food Biosciences, Reading, UK

Yeasts are currently identified by molecular biological methods that arerelatively slow, labour-intensive and require considerable technical exper-tise. Although whole-organism fingerprinting by physicochemical spectro-scopic methods (e. g., pyrolysis-mass spectrometry, FTIR) is possible,cumbersome data analysis and the need for skilled interpretation of spec-tra have precluded adoption of these methods for rapid identification ofyeasts.

Matrix-assisted laser desorption/ionization time of flight mass spec-trometry (MALDI-TOF-MS) was investigated as a method for the rapididentification of yeasts. Characteristic mass spectra were not obtained bydirect analysis. Following pretreatment of yeast samples with a cell walldigesting enzyme (lyticase), however, distinct and reproducible massspectra over the m/z range 2,000 to 16,000 were obtained by MALDI-TOF-MS. Using an optimised procedure, characteristic mass spectra that dis-tinguished between Candida spp and between strains of Saccharomycescerevisiae were produced. The approach offers the potential for rapid andsimple identification of yeasts in clinical diagnosis and in the fermentationindustries.




http://ww

w.m

cponline.org/D

ownloaded from


A.24Oxidative Protein Modifications andAge-related Vision LossJohn W. Crabb1, Xiaorong Gu1,Kutralanathan Renganathan1, Masaru Miyagi1,Quteba Ebrahem1, Bela Anand-Apte1, Stanely L. Hazen1,Daniel T. Organisciak2, Robert G. Salomon3,and Joe G. Hollyfield1

Cleveland Clinic Foundation, Cleveland OH; 2Wright StateUniversity, Dayton OH; 3Case Western Reserve University,Cleveland OH

Age-related macular degeneration (AMD) is the most common cause oflegal blindness in the elderly population of developed countries. Bothgenetic and environmental factors contribute to the disease however thecause of AMD is unknown and presently there are no cures. We hypoth-esize that similar mechanisms of oxidative damage are involved in AMDand retinal light damage and are identifying proteins and protein chemicalmodifications associated with AMD and light damaged rat retina as anapproach to defining these pathways. Major risk factors for developingAMD are extracellular deposits termed drusen, which accumulate with agebeneath the retinal pigment epithelium on Bruchs membrane. The pro-gression of AMD might be slowed or halted if the formation of drusencould be modulated. Liquid chromatography tandem mass spectrometric(LC MS/MS) analyses of drusen preparations from 18 normal humandonors and 5 AMD donors identified 129 proteins [2002 Proc Natl AcadSci USA 99, 14682]. Immunocytochemical studies have thus far localizedabout 16% of these proteins in drusen. TIMP3, clusterin, vitronectin, andserum albumin were the most common proteins observed in normal donordrusen while crystallin was detected more frequently in AMD drusen.Interestingly, crystallins also appear to be more abundant in rat retinafollowing light damage [2003 Exp Eye Res 76, 131]. Oxidative proteinmodifications identified in AMD tissues include apparent crosslinked spe-cies of TIMP3 and vitronectin, carboxymethyl lysine and carboxyethylpyrrole (CEP) protein adducts. CEP adducts are uniquely generated fromthe oxidation of docosahexaenoate-containing lipids and by Western anal-ysis, are more abundant in AMD than in normal Bruchs membrane. Latestage AMD, with the most severe vision loss, involves neovascularizationwhere blood vessels grow from the underlying choroid through Bruchsmembrane into the retina. Our recent results demonstrate that CEP ad-ducts stimulate angiogenesis in vivo in chorioallantoic membrane andcorneal implant assays. CEP immunoreactivity and CEP autoantibody titerare also significantly elevated in plasma from AMD donors relative to thatfrom age-matched normal donors, and may be of diagnostic utility asbiomarkers for predicting AMD susceptibility. Oxidative protein modifica-tions identified in rat retina following intense in vivo light exposure includeCEP adducts, argpyrimidine and nitrotyrosine [2002 Mol Cell Proteomics1, 293]. These data link oxidative injury with AMD and retinal light damageand support a role for oxidative protein modifications in their respectivemechanisms of pathogenesis.

A.25A New Approach to Profile OvarianCancer Serum Using FunctionalizedMagnetic Beads, AnchorChip™Technology, MALDI-TOF MS, MALDI-TOF/TOF MS and BioinformaticsSau-Mei Leung1 and Samuel C. Mok2

1Clincial Proteomics, Bruker Daltonics Inc., Billerica, MA; 2BWH,Harvard Medical School, Boston, MA

Most cases of ovarian cancers are detected at late stages and are rarelycurable. There is an urgent need to develop early detection assays with thehope of improving survival. Multiple strategies have been applied to iden-tify differentially expressed proteins in serum samples of ovarian cancerpatients and have already provided promising results. However, there arestill a limited number of proteomic platforms which can be used to gen-erate reliable protein profiles from serum samples, which can help us toidentify early diagnostic markers for the disease. We have developed anew, integrated method for serum profiling called ClinProt™ Solutions.This tool set utilizes: (1) magnetic beads and AnchorChip™ technology forsample fractionation and preparation before mass spectrometry (MS)analysis, (2) Matrix Assisted Laser Desorption Ionization-Time Of Flight(MALDI-TOF) and MALDI-TOF/TOF MS to generate protein profiles in bothlinear and reflector modes, (3) MALDI-TOF/TOF MS to identify low molec-ular weight peaks using the same sample spot, and (4) bioinformatics(ClinProTools™ software) for data reduction, mining, and visualization.Our results show that this new approach is reproducible and the hydro-phobic magnetic beads are able to fractionate human serum. In addition,the AnchorChip technology provided high sensitivity in MS detection byconcentrating the matrix/fractionated serum samples to a 600um spot. Wealso found that both linear and reflector modes in MALDI-TOF analysisoffer different advantages in protein profiling in terms of number of peaksdetected, resolution and mass accuracy. We have demonstrated thatusing this new approach, differential protein profiles can be generatedfrom serum samples obtained from stage I ovarian cancer patients vs.age-matched healthy women. We will also describe the general profilingstrategies using this new integrated method.




http://ww

w.m

cponline.org/D

ownloaded from


A.26Mass Spectrometric Analysis of IntactShewanella oneidenis Proteins UsingCapillary RPLC/FTICR and CIEF/FTICRSeonghee Ahn, Bogdan Bogdanov, Andrey N. Vilkov,Dae-Ho Shin, David Simpson, Rui Zhao, Ljiljana Pasa-Tolic,Mary Lipton, and Richard D. Smith

Environmental Molecular Sciences Laboratory, Pacific NorthwestNational Labotatory, Richland, WA

The identification of proteins at the peptide level has been widely used inhigh-throughput mass spectrometric proteomic analysis. However, meth-ods based on tryptically digested proteins (i. e., bottom-up) are proven tobe ineffective for identifying protein modifications. Analysis of the intactproteins (i. e., top-down) can provide information of co- or post-transla-tional modifications, but such analysis of complex protein mixtures hasbeen a much greater experimental challenge for both mass spectrometricand separation techniques. In this study, we demonstrate the method anddata processing technique for the analysis of intact proteins using bothcapillary reverse phase liquid chromatography (RPLC) and capillary iso-electric focusing (CIEF) coupled on-line with Fourier transform ion cyclo-tron resonance (FTICR) mass spectrometry.

Cells from Shewanella oneidenis were lysed by beating with 0.1mmzirconia/silica beads, centrifuged, and the cytosol drawn off from thepellets after centrifugation. A portion of this complex mixture was trypti-cally digested and analyzed by LC-MS/MS (using an ion trap), while theremainder was injected onto a size exclusion column (SEC) or a C-4column (10 �g particle with 300 Å pore size) for fractionation. The LC-MS/MS data were processed and analyzed using the SEQUEST programto generate a potential mass tag (PMT) database. A portion of eachfraction generated by either SEC or C-4 was also tryptically digested andanalyzed using LC-MS/MS and SEQUEST to generate correspondingPMT databases. The remainder of each SEC fraction was analyzed bycapillary RPLC/FTICR. The capillary RPLC with 80 cm columns packedwith C-5 bonded particles (5 �g particles with 300 Å pore size) providedhigh-efficiency separation at pressure up to 10,000 psi. The residue ofeach C-4 fraction was analyzed by CIEF/FTICR. The columns for CIEFwere coated with 5 percent hydropropyl cellulose and 1 percent pharma-lyte was added to the fractionated samples. Mass spectra were acquiredusing the 7-Tesla FTICR mass spectrometer coupled with capillary RPLCor CIEF. This instrument is equipped with three serial quadrupoles, includ-ing two consecutive high-quality selection quadrupoles and an accumu-lation quadrupole, and provides a short duty cycle and high mass meas-urement accuracy (MMA).

Comparison with the data obtained using non-fractionated sample in-dicated a significant increase in the number of proteins detected when thesample is prefractionated through a combination of bottom-up and top-down methods.

A.27Pathogenic Bacterial Profiling andCataloguing by LC MALDI Tandem MSAnalysisSanjeev Bhardwaj and Melanie Lin

Applied Biosystems, Framingham, MA

Mass spectrometry has become the technology of choice for approachingthe structural proteomics arena and for addressing challenges in drugdiscovery and diagnostics area. With the advent of bioterrorism, it hasbecome imperative to develop applications to quickly diagnose and coun-ter those threats. One application would be to develop a library of peptideand MS/MS fingerprints of the pathogenic bacterial proteins and cata-logue them for diagnostics purpose by mass spectrometry. In the case ofbioterrorist activity, the type of pathogen could then be identifiedthrough library searching. The application would help to reduce thediagnosis time tremendously and thus enable quicker counter-responseto such activities.

The analyses of complex proteins such as bacterial proteins have beenlimited in the past by lack of tools that can handle such complexity andidentify especially low abundance proteins. The newly developed LC-MALDI approach coupled with tandem MS makes it possible to overcomesuch challenges. The 4700 Proteomics Analyzer with TOF-TOF™ ionoptics used in this study not only provides high sensitivity associated withMALDI but also acquires automated MS/MS of selected ions in a highthroughput manner. In this study, the entire pathogenic bacterial cellproteins were digested and analyzed using LC-MALDI MS/MS to deter-mine protein identification efficiency and the feasibility of such workflowsin dealing with real life samples in a timely manner. The total protein mixwas reduced, alkylated and digested prior to the analysis. The sample wasinjected onto a nano-LC column with 5 second fractions deposited in a24x24 array on two MALDI targets. MS/MS analyses were then performedusing an intelligent algorithm for dynamic exclusion precursor ion selec-tion. Protein identifications were achieved through database searching bycombining all MS/MS from this sample using GPS Explorer™ softwarethat incorporates the MASCOT search engine.

The work will demonstrate that LC-MALDI analysis can identify hun-dreds of proteins from a cell lysate by reverse phase separation in a highthroughput manner. Preliminary data from the LC MALDI workflow on E.coli cell lysate have successfully resulted in identification of hundreds ofproteins. The ultimate goal of this study is to generate MS/MS fingerprintsof the pathogenic bacterial proteins from all strains and build a library thatcan be used for rapid identification of bioterrorism agents thereby reduc-ing the time to deploy countermeasures. The future work would involveanalysis of several strains of pathogenic bacteria and library constructionto enable achievement of the broader goals.

Acknowledgement: We would like to thank Dr. Hercules Moura and Dr.John R. Barr from ERAT/DLS, Center for Disease Control, Georgia forproviding us with the pathogenic bacterial samples.




http://ww

w.m

cponline.org/D

ownloaded from


A.28LC/MS/MS Analysis of Post-translationalModifications in Chicken Myelin BasicProteins with Trypsin and EndoproteinaseGlu-C DigestionsJeongkwon Kim1, David G. Camp, II1, Harold Udseth1,Richard D. Smith1, and Robert Zand2

1Environmental Molecular Sciences Laboratory, Pacific NorthwestNational Laboratory, Richland, WA; 2Biophysics Research Division,Department of Biological Chemistry, The University of Michigan,Ann Arbor, MI

In the central nervous system, myelin membrane contains a major proteinthat helps to maintain the structure of the myelin membrane. This proteinknown as myelin basic protein (MBP) is an antigenic protein that is thoughtto be involved as the autoimmune agent in multiple sclerosis. MBP main-tains the integrity of the myelin sheath that wraps around the nerve axonand facilitates the velocity of nerve impulse conduction. MBP in mamma-lian myelin is known to possess post-translational modifications (PTMs),such as phosphorylation, methylation, deamidation and citrullination. Inthe present study, three charge isomers (C1 through C3) were purifiedfrom chicken brain MBP. They were analyzed using capillary liquid chro-matography combined with mass spectrometry and their PTMs wereidentified. Identification of PMTs on MBP is important because it canprovide information on the role of MBP in the etiology of multiple sclerosisin mammalian species as well as the evolutionary path from non-mam-malian vertebrates to higher level mammalian animals.

Sequence coverage of 100% for each MBP charge isomer wasachieved from the combination of tryptic and endoproteinase Glu-C di-gestions. A high abundance of basic amino acid residues in the proteinsequence results in small tryptic digested peptides, which do not retain onthe column. Peptides from endoproteinase Glu-C digestion are sufficientlylong to be retained on the C18 column, eluted by the acetonitrile gradient,and subsequently detected by the mass spectrometer.

MBP C1 has methionine oxidation at methionines 14 and 100, and amixture of mono- and di-methylations at arginine 105. MBP C2 has me-thionine oxidation at methionine 100, a mixture of mono- and di-methy-lations at arginine 105, and phosphorylation at serines 33, 96, and 168.MBP C3 has methionine oxidation at methionines 14 and 100, a mixture ofmono- and di-methylations at arginine 105, phosphorylation at serines 33,96, 113, 164 and 168, citrulline formation at arginine 41, and deamidationat glutamine 146.

Support of this work by the National Multiple Sclerosis Society (Grant #RG 3167-A-1) to Robert Zand is gratefully acknowledged. Pacific North-west National Laboratory is operated by Battelle Memorial Institute for theU.S. Department of Energy under contract DE-AC06–76RLO 1830.

A.29Atmospheric Pressure IR Ionization fromSolutions (AP IRIS) for Analysis ofComplex CarbohydratesNelli I. Taranenko1, Philip Tan1, Vladimir Doroshenko1, andBrian Musselman2

1MassTech, Inc., Columbia, MD; 2SciMarket Strategies, Inc.,Melrose, MA

Glycosylation, the covalent attachment of saccharides to specific aminoacid residues, is one of the most common post-translational modificationsin proteins. Among all existing technologies, including three-dimensionalchromatographic techniques, mass spectrometry has facilitated oligosac-charide analysis and has become the method of choice for rapid detec-tion/identification of individual glycans.

The goal of this study was to extend the success of AP-MALDI massspectrometry from its established capabilities in proteomics applicationsto oligosaccharide analysis. Conserved trimannosyl core-substituted withfucose (M3N2F Glycan, neutrally charged); Asialo-, agalacto, biantennary(NGA2 Glycan, neutrally charged), Oligomannose 6 (Man 6, neutrallycharged) and Mono-sialylated-, galactosylated, biantennary core-substi-tuted with fucose (A1F Glycan, negatively charged), carbohydrate stand-ards were chosen for this investigation and were obtained from PROzymecompany (San Leandro, CA, USA). A comparison between AP infraredlaser ionization from solution (AP IRIS) with UV AP-MALDI for the analysisof oligosaccharides will be presented in full MS spectra and in MS/MSstructural spectra.

All experiments were carried out on a Finnigan LCQ DECA XP (SanJose, CA, USA) ion-trap mass spectrometer equipped with an AP/MALDIionization source (MassTech Inc., Columbia, MD, USA). The heated cap-illary was set at 280 °C. The instrument was operated in the positive ornegative ion modes with the conversion dynode voltage at 15 kV. InMS/MS experiments, the standard resonance excitation method was em-ployed. The setup for AP-IRIS experiments essentially substituted the UVlaser with an IR laser and rotated the sample target plate so that it wasparallel with the sampling capillary axis. Our AP-IRIS utilized an in-housebuilt Yb:YAG-pumped optical parametric oscillator (OPO) infrared laser toprovide 30 ns pulses with a frequency of 5Hz. The wavelength of thepulses can be tuned in the region 2.8–3.1 micrometers, while the energyper pulse remains 0.4–0.5 mJ.

Results showed that the optimal wavelength for aqueous solutions ofthe carbohydrate standards was around 2.94 micrometers. The carbohy-drate standards were detected mainly as (M � Na)� ions at low pmolesensitivity. Using only positive ionization mode we were able to analyzemixtures of both neutral and acidic oligosaccharides. The collision in-duced dissociation of the protonated molecule for each of the glycansyielded easily interpretable spectra. Every major ion present in the spectrawas linked to structural features. Based on this data it was shown thatAP-MALDI can play a critical role in successful analysis of complexcarbohydrates.




http://ww

w.m

cponline.org/D

ownloaded from


A.30Toward the Automated Identification ofPhosphorylation Sites in Proteomics byLC-MS/MSPeter Juhasz, Brian Williamson, Jason Marchese, and SteveMartin

Applied Biosystems, Framingham, MA

Phosphorylation of proteins is perhaps the most widely studied post-translational modification due to its importance in numerous cellular pro-cesses. Automated determination of phosphorylation sites on the pro-teome scale is greatly hampered by the poor detection sensitivity ofphosphopeptides in complex peptide mixtures analyzed by LC-MS/MS.One approach to overcome this difficulty employs affinity enrichment ofphosphopeptides through metal chelators (IMAC) and uses LC-MS/MS toidentify phopshorylation sites. The moderate efficiency, specificity, andreproducibility of IMAC fractionation are still preventing routine low-leveldetermination of phosphorylation. Another approach utilizes the combina-tion of precursor ion scanning on a triple quadrupole mass spectrometerto detect phosphorylated components with a second MS/MS analysis ona suitable instrument to obtain high sensitivity product ion spectra forsequence information. On the scale of proteomic experiments, both ap-proaches suffer from the increased probability of false identifications ifvariable modifications of Ser, Thr, and Tyr have to be considered with largedatabases.

The presented approach uses a linear quadrupole ion trap instrument(QTRAP, Applied Biosystems) for the selective identification of phospho-rylated proteins in an LC-MS experiment. This platform features bothsensitive precursor scanning and product ion scanning. First, the charac-teristic PO3

- fragment ion is detected at m/z 79. Then polarity switching,enhanced resolution scan to obtain the accurate mass of the peptide, andan MS/MS sequencing scan follow to complete the analysis cycle. Thiscycle can be executed in less than 5 s rendering this approach compatiblewith the time scale of HPLC elution. LC-MS analysis of protein standardssuch a �-casein, ovalbumin, fetuin from in-solution and in-gel digestsyielded the determination of known phosphorylation sites but also a fewothers not reported so far. Sensitivity levels of 10–20 fmole from solutionphase digests could be reached.

To demonstrate the feasibility of proteome level experiments, cell ly-sates of wt. yeast and purified nuclei of human fibroblast cells wereapplied to 1D gels. Selected gelbands were excised, digested and 50% ofthe sample was analyzed first by MALDI LC-MS/MS (AB4700, AppliedBiosystems). The identified sets of proteins were compiled into a search-able FASTA files. The second 50% of sample was analyzed on the QTRAPin an automated LC-MS experiment as described above. Product ionspectra of phosphopeptides were generated and searched against thepreviously generated FASTA files using Mascot. Preliminary experimentsidentified a novel phosphorylation site (Ser-1157) on yeast Acetyl CoASynthetase: a protein known to be regulated through phosphorylation.Analysis of the nuclear proteins is also expected to reveal a number ofnovel phosphorylation sites.

A.31Use of Automated MDLC with TimeFiltered Exclusion for the Identification ofDNA Binding Proteins in E. coliChristie L. Hunter and Lydia Nuwaysir

Applied Biosystems, Foster City, CA

2D Gel electrophoresis coupled to mass spectrometry is regarded as apowerful tool in the separation and identification of complex protein sam-ples. Despite its high resolving power the technique has limitations whenit comes to the separation and identification of membrane and low abun-dance proteins. Multi-dimensional chromatography (MDLC) coupled tomass spectrometry provides an alternative to this technique, and allowsaccess to these other classes of proteins. In the most popular form of thistechnique, protein mixtures are first digested and the resulting peptidesare loaded onto a cation exchange chromatography column for fraction-ation either off or on line via a salt gradient. These fractions are then furtherseparated by low flow reverse phase LC coupled to a mass spectrometer.

MDLC in combination with a quadrupole-time of flight mass spectrom-eter was used to investigate a subset of E. coli proteins, the DNA bindingproteins. These were isolated using a DNA affinity column and eluted intotwo salt fractions using 0.4M and 1M NaCl. These two fractions were thenstudied by MDLC-MS. Multiple MDLC runs were performed using timefiltered exclusion lists created from previous protein identifications fromPro ID software to obtain more MS/MS spectra on the very low abundancecomponents of the sample. Over 80 proteins were found in the high saltfraction and over 140 proteins were found in the low salt fraction, with highconfidence. Codon bias calculations were done on the protein hits aftereach analysis to investigate the utility of performing multiple runs with timefiltered exclusion lists to find a greater number of low abundance proteins.Pro ID Software can be used with Celera Discovery System protein se-quence databases. Following identification, proteins were further analyzedfor nucleic acid binding domains as well as other motifs, domains, andpatterns using sequence analysis software (helixturnhelix, prositesearch)in the Celera Discovery System. Additionally, the molecular functions andbiological processes of identified proteins were investigated using thePanther Protein Function-Family Browser, also in the Celera DiscoverySystem.




http://ww

w.m

cponline.org/D

ownloaded from


A.32Singlet Oxygen-induced Oxidation ofProteins: Evidence for Non-catalyticProduction of Hydrogen PeroxideJunlong Shao1, Jack Lancaster2, Marion Kirk3, andStephen Barnes1,2,3

1Purdue-UAB Botanicals Center; 2Center for Free Radical Biology;3Mass Spectrometry Shared Facility; University of Alabama,Birmingham, AL

Singlet molecular oxygen is a highly reactive oxygen species (ROS), whichparticipates in a variety of physiological and pathological processes, suchas oxidative stress, radiation damage and aging. In vivo, singlet oxygencan oxidize tryptophan, histidine and methionine, initiate membrane lipidperoxidation and lead to protein modifications and inactivation of manyenzymes. Singlet oxygen is generated by NADP oxidases as well as byultraviolet light. We have investigated the hypothesis that singlet oxygengenerates supramolar amounts of ROS by reacting with multiple aminoacid residues of proteins. This was tested by exposing solutions of se-lected proteins to ultraviolet light and other sources of singlet oxygen. Thetreated protein samples were analyzed by electrospray ionization on aMicromass Q-Tof3 and their molecular weights determined by MaxEnt.Control and treated proteins were cleaved with specific proteases andsubjected to MALDI-TOF mass spectrometry and tandem mass spectrom-etry following nanoLC analysis.

In vitro mass spectrometric investigations of protein oxidations initiatedby singlet oxygen revealed multi-oxygen oxidation states (i.e., multiplepeaks differing in mass by 16 Da) of a variety of proteins (beta-lactoglob-ulin A, beta-galactosidase, mouse IgA k and rat IgG1 l). For beta-lacto-globulin A, exposure to UV light for 60 min led to the sequential addition ofsix oxygen atoms. Minor amounts of even higher numbers of addedoxygen atoms were observed. In addition to ultraviolet light, two othersinglet oxygen sources were used (visible light exposure with photosen-sitizer and the endoperoxide of NDP) in these studies. Interestingly, all theabove radical reactions can produce H2O2. Some amino acids have beenstudied under the same conditions as the proteins in order to elucidate themechanisms of these reactions. Furthermore, the trypsin digestions of theoxidized proteins were analyzed by LC/MS/MS to locate the oxidizedresidues. We conclude that supramolar production of ROS by certainproteins occurs by a chemical reaction of proteins with singlet oxygen,rather than a catalytic event. These data suggest experimental models forthe chemical consequences of protein oxidation in the aging process andin tissues naturally exposed to UV light (the eyes and the skin), as well asinvestigation of the roles of polyphenols as antioxidants in these systems.

A.33Chemical Adduction Sites in Cytochromec Characterized by Mass SpectrometryMaria D. Person, Samy L. Habib, Terrence J. Monks, andSerrine S. Lau

The University of Texas at Austin, Division of Toxicology, Austin, TX

A challenging and important area of research in functional proteomics isthe detection of chemical-induced protein modifications via covalent ad-duction. Such aberrant post-translational modifications may alter thestructure and function of the modified protein with the potential for inap-propriate modulation of cell signaling pathways. To investigate whether ornot “electrophile binding motifs” exist in a given protein and recognizevarious environmental chemicals, we examined the covalent adduction ofcytochrome c, a key protein involved in apoptosis. Cytochrome c under-goes modification by reaction in vitro with the environmental chemicalbenzoquinone and its toxic metabolite benzoquinone-glutathione conju-gate, and the alkylating agent iodoacetamide. Using comparativeMALDI-MS and ESI-MS and targeted ESI-MS/MS and MALDI-PSD, wehave identified the specific sites on the protein targeted preferentially bythese agents. Enzymatic digests of modified proteins with trypsin andchymotrypsin are used to increase protein coverage and determine thesites of adduction. A novel cyclic di-quinone species is the predominantspecies produced during quinone adduction, and the benzoquinone-glu-tathione conjugate undergoes an elimination/cyclization reaction on cyto-chrome c at basic pH. The cyclic products are primarily produced at twoseparate sites on the protein. In contrast, iodoacetamide and benzoqui-none-glutathione conjugate at acidic pH form non-cyclic adducts boundto cytochrome c at multiple sites, including the sites previously identified.The Scoring ALgorithm for Spectral Analysis (SALSA) provides a comple-mentary method for detection of low abundance quinone-thioether ad-ducts using characteristic neutral loss ions. Lysine and histidine residuesare targeted by the reactive chemicals, with solvent exposure, tertiarystructure and local pKa all playing important roles in the reactivity ofindividual sites. (GM39338, ES07784)




http://ww

w.m

cponline.org/D

ownloaded from


A.34Automated Chip-based NanoelectrosprayMass Spectrometry for the QuantitativeDetermination of NoncovalentProtein-ligand Binding InteractionsSheng Zhang, Colleen K. Van Pelt, and Wayne C. Muster

Advion BioSciences, Inc., Ithaca, NY

Noncovalent protein-ligand interaction investigations by electrospray ion-ization mass spectrometry (ESI/MS) are of interest because of their im-portance to molecular recognition and combinatory ligand library search-ing. ESI/MS has many advantages including high sensitivity, speed ofanalysis, capability of obtaining stoichiometric information and the abilityto identify unknown molecules. The goal of this work was to determine ifan automated nanoelectrospray system coupled to a mass spectrometercould reliably determine noncovalent interactions as well as quantitativelydetermine the absolute dissociation constants for protein target andligands.

The NanoMate™ 100 and ESI Chip™, an automated nanoelectrospraysystem, have been developed to improve the efficiency and quality ofESI/MS methods. Advantages of the system include low sample con-sumption, conservation of sample not consumed in the analysis, one-timespray optimization, enhanced spray stability, and no carryover. The sys-tem aspirates samples from a 96-well microtiter plate using disposable,conductive pipette tips, and then delivers samples to the inlet of the ESIChip, a microarray of ESI nozzles microfabricated in silicon. A pressureseal is formed around a through-chip channel at one nozzle and nano-electrospray is initiated by applying a spray voltage and psi pressure to thesample in the pipette tip.

Two protein-ligand systems were used for quantitative determination ofnoncovalent binding interactions. One was RNase complexed with cyti-dine ligands (CMP and CTP), which is a well characterized model systemwith published dissociation constant information for comparison and val-idation. The other example was an endocellulase complexed with fouroligosaccharide ligands: cellotriose(G3), cellotetraose(G4), cellopentao-se(G5) and cellohexaose(G6). The titration experiments were performedusing a constant protein concentration with varying ligand concentrations.All reactions were incubated at room temperature for 10 minutes prior tonanoESI/MS analysis using a NanoMate coupled with a Q-TOF MS. Theresults demonstrate automated nanoelectrospray can be used for rapidscreening of potential candidates in drug discovery programs.

A.35Strategies for MS Westerns Using aQuadrupole Time-of-Flight MSDanie M. Schlatzer, Joanna Krise, Kevin Blackburn, andArthur Moseley

GlaxoSmithKline, Research Triangle Park, NC

The recent introduction of targeted protein identification methods (i.e., MSWesterns, Arnott et al., Molecular and Cellular Proteomics 1:148–156,2002) have enabled new hypothesis driven approaches for validatingbiological findings in the absence of suitable antibodies for traditionalWestern Blots. In the present work, we have evaluated and optimizedtargeted protein identification strategies using a quadrupole time-of-flightmass spectrometer (Q-Tof). Utilizing the high mass accuracy and resolu-tion of the Q-Tof, we have developed methodologies that are highlysensitive and selective for identifying proteins from complex biologicalsamples. Applying this approach, we demonstrate limits of detection of�100 fmoles for a standard protein spiked into a complex background.This would correspond to a �1500 copy number protein from a samplecontaining 5 � 107 cells. In addition, the use of multi-dimensional chro-matography and/or multiple MS survey ranges has enabled multiplexedanalyses for targeting multiple proteins in the same analysis. Data will alsobe presented from isotope coding experiments for targeted quantitativecomparisons.

A.36Using Comparative Proteomics andFunctional Genomics to UnderstandCytokinesisAhna R. Skop1, Honbin Liu2, John Yates2,Barbara J. Meyer1, and Rebecca Heald3

1University of California, Berkeley, and Howard Hughes MedicalInstitute; 2The Scripps Research Institute, La Jolla, CA; 3Universityof California, Berkeley

Understanding how an exact copy of the genome is transmitted during celldivision is a key question in biology, yet systematic identification andfunctional characterization of the relevant proteins has been lacking. Toobtain an enriched source of cell division proteins, we isolated mammalianmidbodies and identified 159 midbody proteins by tandem liquid chroma-tography and tandem mass spectrometry (LC/LC/MS/MS). To assessfunction, we inactivated the homologues of the mammalian proteins in C.elegans using RNA-mediated interference (RNAi), and found that 88%played a role in either mitotic or meiotic cell division events. The majorityof the midbody proteins functioned in membrane-cytoskeletal dynamics,with their disruption resulting in a high percentage of cytokinesis defects(76%), as well as neuronal defects (16%). The extensive conservation ofthe proteins identified points to common and ancient mechanisms medi-ating cell division and membrane dynamics, all of which are critical inhuman development and defective in cancer and/or neurodegenerativediseases.




http://ww

w.m

cponline.org/D

ownloaded from


A.37Multidimensional Liquid PhaseSeparations of Intact Proteins as anAlternative to 2D Gel Electrophoresis forProteomicsAlex Apffel, Hongfeng Yin, Tom Vandegoor,and Kevin Killeen

Agilent Laboratories, Palo Alto, CA

In the field of expression proteomics, the dominant separation technologyhas been two dimensional gel electrophoresis (2DGE) frequently coupledwith mass spectrometric identification methods. Although an extremelypowerful separation technique, 2DGE suffers from a number of drawbacksincluding lack of speed and automation, poor reproducibility and quanti-tation and fundamental limitations in linearity resulting in a bias towardshighly expressed proteins. Alternative approaches based on multidimen-sional HPLC separation of proteolytic digests of complex samples (e.g.,Mudpit) have emerged combining nanoscale reversed phase separationsof strong cation exchange fractions. Although these approaches havedemonstrated utility, the complexity of the resulting mixture represents aconsiderable chromatographic challenge. Furthermore, pooling the pro-teolytic fragments from a large number of proteins eliminates any associ-ation of a given peptide with its parent proteins and thus configurations ofmultiple posttranslational modifications.

In an effort to overcome the limitations of existing techniques, we havebeen evaluating the use of multidimensional liquid phase separation ofintact proteins as an alternative. In this approach, fractions from a firstdimension are collected and re-analyzed by a second, orthogonal sepa-ration mechanism. As 1st dimension modes, we have evaluated anionexchange chromatography, size exclusion chromatography, conventionaland gradient chromatofocusing and preparative isolectric focusing (Roto-for). As a 2nd dimension, high speed separations are utilized using a 5�m300A Poroshell 300SB-C3 material at high temperatures (60 °C). Thissuperficially porous material allows rapid diffusion of large molecularweight proteins resulting in reversed phase cycle times of 5–10 minutes/fraction. The combinations of these chromatographic modes are largelyorthogonal, utilize compatible mobile phases and yield resolution of 1000proteins in a single analysis. Fractions eluting from the 2nd dimension arecollected, digested and analyzed by mass spectrometry. The use of rapidnanospray LC-MS/MS, utilizes a 3rd orthogonal dimension of separationof the initial complex sample, while maintaining intact protein informationwith excellent sensitivity.

A.38

PrepTips: A New Method for Cleaning andConcentrating Biological Samples Prior toMass Spectrometric AnalysisJennifer R. Krone1, David Miller1, Issa Isaac1, CecilyPeriana2, Mike Pisano3, and Ron Sostek3

1Genomic Solutions, Ann Arbor, MI; 2Harvard Apparatus;3Proteomic Research Services, Ann Arbor, MI

PrepTip is a new method for cleaning and concentrating proteins andother biological samples for further instrumental analysis. PrepTip’sunique feature is that the interior walls of the tip are coated with thesample-binding material. This interior coating technology provides Prep-Tip with both speed and reliability. Since the tip opening is not plugged bythe interior coating process, the sample can flow freely through the open-ing. PrepTip can be used to clean or concentrate any biological samplesince the interior walls of the tip can be coated with a variety of bindingmedia. Due to its unique features, sample preparation with PrepTip is veryfast, simple and highly effective.

Bovine serum albumin was digested with Trypsin using a GenomicSolutions Investigator ProPrep robotic station. Peptides from the trypticdigests were isolated and concentrated using Harvard Apparatus Prep-Tips and Millipore ZipTips. The samples were then spotted to a MALDItarget and analyzed using an ABI Voyager DE-STR. Protein identificationand coverage maps were generated using ProFound.

We will present data from our PrepTips and ZipTips peptide bindingstudy. The PrepTips binding capacities equal, and in some cases exceed,that of ZipTips. PrepTips also give better results when considering proteincoverage. PrepTips will be shown to have many advantages over ZipTips.The unique PrepTips interior coating matrix means no more plugging ofthe tip opening. This will allow easy, rapid flow of sample due to theunobstructed tip opening. PrepTips are also very effective for small sam-ples (even with volumes less than 1 �l). Most importantly the PrepTips willbe shown to be mass spectrometry amenable.




http://ww

w.m

cponline.org/D

ownloaded from


A.39The Role of TMT1 in a Common RepairSystem for Methylene Addition PathwayByproducts: The EI GC/MS Identificationof 3-Isopropylmalate as a Novel Tmt1pSubstrateDarren S. Dumlao1, Jonathan E. Katz1,Jacob I. Wasserman1, Michael E. Jung1, Kym F. Faull2, andSteven G. Clarke1

1Department of Chemistry and Biochemistry, UCLA; 2Department ofPsychiatric and Biobehaviorial Sciences, UCLA

Previously, we have shown that the Saccharomyces cerevisiae gene prod-uct, TMT1, and its E. coli homologue, Tam, recognize and methylatetrans-aconitate, a thermodynamically favored and spontanously formedracemer of cis-aconitate and an inhibitor of the citric acid cycle. We havealso shown that in S. cerevisiae there is an alternate major substrate ofTmt1p that is not trans-aconitate.

Here we present the methodologies that we used to partially purify andultimately identify the alternate substrate as 3-isopropylmalate. Wildtypeand TMT1 knockout yeast cells were cultured with the radiolabeled methyldonor S-adenosyl[3H-methyl]methionine and cell lysates were preparedand chromatographed by HPLC. Electron Impact GC/MS spectra wereobtained for fractions that had an abundant radioactive peak present inwildtype extracts that was absent in TMT1 knockout extracts. We thendeveloped custom software to identify spectral differences. Based onpredictions from our spectra, we synthesized 3-isopropylmalate and con-firmed that this was our alternate substrate based on GC elution time andEI spectral similarities.

Kinetic assays were performed to confirm the in vitro activity of theenzyme towards 3-isopropylmalate. TMT1 was found to have similar ac-tivities towards both trans-aconitate (Km 0.055 mM, Vmax 72.44 nmol/min/mg) and 3-isopropylmalate (Km 0.0118 mM, Vmax 67.75nmol/min/mg).

3-Isopropylmalate is an intermediate in a series of reactions that per-form methylene addition to an alpha-keto acid in leucine biosynthesis;cis-aconitate is an intermediate in methylene addtion to an alpha-keto acidthat occurs in the citric acid cycle. We currently believe that TMT1 mightact as a “molecular guardian” over many of these types of pathways bymethylating non-biologically active, spontaneously formed deleteriousisomers of intermediates in these pathways. We are currently exploringwhich isomers of the various pathway intermediates are most recognizedby Tmt1p.

A.40Intact Mass Proteomics of BiologicalMembranesJulian P. Whitelegge1, Rodrigo Aguilera1, Alek Dooley1,Stephen Gomez1, Jarlath Nally2, Mike Lovett2,Wim Vermaas3, and Kym F. Faull1

1The Pasarow Mass Spectrometry Laboratory, UCLA; 2Departmentof Medicine, UCLA; 3School of Life Sciences, Arizona StateUniversity

Integral membrane proteins function to drive, catalyze, transport andtransduce signals in many vital cellular processes making them populardrug targets for the pharmaceutical industry. Since a well-resolved massspectrum of a protein defines the native covalent state of the correspond-ing gene’s product, as well as associated heterogeneity, it is a very usefulmeasurement in proteomics. Over the past few years we have developeda suite of techniques for analysis of intact integral membrane proteinsusing electrospray-ionization mass spectrometry. The reverse-phase andsize-exclusion liquid chromatography mass spectrometry systems (LC-MS�) employed provide sufficient resolution to analyze samples of mod-erate complexity and range of abundance. This has been illustrated by astudy demonstrating full subunit coverage of the cytochrome b6f complexfrom photosynthetic membranes (Whitelegge et al. (2002) Mol. Cell. Pro-teomics 1, 816–827). The intact masses that were measured for cyto-chrome b led us to report that this four trans-membrane helix protein wasmodified with a heme molecule, a conclusion that was recently confirmedby a high-resolution X-ray crystal structure of the complex. When outermembrane preparations from the spirochete Leptospira were analyzed byLC-MS�, information complementary to that from 2D-gel analyses wasobtained; intact protein mass accuracy was dramatically better, proteomecoverage was extended and heterogeneity of lipid modification was quan-tified. To extend the chromatographic technologies toward full membraneprotein coverage in the context of a full proteomics project, a seconddimension of chromatography has been applied prior to our aqueous/organic LC-MS� systems. This 2D chromatography system will be com-pared to a 2D-gel based study of the membranes of the photosyntheticprokaryote Synechocystis sp. PCC 6803 both in terms of proteome cov-erage and fidelity of the analytical procedures toward preserving the nativecovalent state of proteins after extraction from cells.




http://ww

w.m

cponline.org/D

ownloaded from


Date post:	12-Sep-2021
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

2.2 2.3 Free-Flow Electrophoresis: A Highly Quantitating Molecular

Documents