Multiplex Analysis

By: Gourab Ray

Research Scholar

Dept. of Biotechnology

Bangalore UniversityUnder Guidance of:Dr. Ravikiran T.Professor, Dept. of Biotechnology,Bangalore University

Agenda

• Multiplex Analysis & Types of Multiplex assays.– Protein Microarray– Antibody Microarray

• Multidimensional Liquid Chromatography– Principle– Types– Applications

Multiplex Analysis

• Multiplex assay is a type of assay that simultaneously measures multiple analyte in a single run/cycle of the assay. It is distinguished from procedures that measure one analyte at a time.

• “Multiplex” refers to those with the highest number of analyte measurements per assay (up to millions) and "low-plex" or "mid-plex" refers to procedures that process fewer (10s to 1000s).

• Widely used in functional genomics experiments that require to detect the state of all biomolecules of a given class (e.g., mRNAs, proteins) within a biological sample.

• To determine the effect of an experimental treatment or the effect of a DNA mutation over all of the biomolecules or pathways in the sample.

Types of Multiplex Assays

Multiplex Assays

Nucleic acid-based

DNA microarray

Serial analysis of gene

expression 

(SAGE)

Protein-based

Protein microarray

Antibody microarray 

Other techniques

Tissue microarray

Cellular microarray

Chemical compound microarray

Protein Microarray• High-throughput method used to track the interactions and

activities of proteins and to determine their function, and determining function on a large scale. Its main advantage that large numbers of proteins can be tracked in parallel.

• Technique is same as DNA Microarray• Developed due to the limitations of using DNA microarrays for

determining gene expression levels in proteomics.• Quantity of mRNA in the cell often doesn’t reflect the

expression levels of the proteins they correspond to. Post-translational modifications, which are often critical for determining protein function, are not visible on DNA microarrays.

• Since it is usually the protein, rather than the mRNA, that has the functional role in cell response, a novel approach was needed.

Protein Microarray• The chip consists of a support surface such as a glass slide,

nitrocellulose membrane, bead, or microtitre plate, to which an array of proteins is fixed.

• Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilized protein emits a fluorescent signal that is read by a laser scanner.

• Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents.

Protein Microarray-Analytical & Functional• Fig a:  Analytical protein microarray. Different types 

of ligands, including antibodies, antigens, DNA or RNA aptamers, carbohydrates or small molecules, with high affinity and specificity, are spotted down onto a derivatized surface. These chips can be used for monitoring protein expression level, protein profiling and clinical diagnostics. Protein samples from two biological states to be compared are separately labelled with red or green fluorescent dyes, mixed, and incubated with the chips. Spots in red or green colour identify an excess of proteins from one state over the other.

• Fig b: Functional protein microarray. Native proteins or peptides are individually purified or synthesized using high-throughput approaches and arrayed onto a suitable surface to form the functional protein microarrays. These chips are used to analyse protein activities, binding properties and post-translational modifications. With the proper detection method, functional protein microarrays can be used to identify the substrates of enzymes of interest. Consequently, this class of chips is particularly useful in drug and drug-target identification and in building biological networks.

Protein Microarray-Methodology• The proteins are arrayed onto a solid surface such as

microscope slides, membranes, beads or microtitre plates.• The chosen solid surface is then covered with a coating that

must serve the simultaneous functions of immobilizing the protein, preventing its denaturation, orienting it in the appropriate direction so that its binding sites are accessible, and providing a hydrophilic environment in which the binding reaction can occur, display minimal non-specific binding in and it needs to be compatible with different detection systems.

• Robotic contact printing or Robotic spotting: robots place large numbers of proteins or their ligands onto a coated solid support in a pre-defined pattern.

• Ink-jetting: a drop-on-demand, non-contact method of dispersing the protein polymers onto the solid surface in the desired pattern.

Protein Microarray-Methodology• The probe labelled with fluorescent dye having affinity for a

specific protein or antigen is applied and allowed for incubation.

• Excess probe is washed off and the microarray is then placed under laser scanning for identification of the spots emitting fluorescent marker.

Protein Microarray-Microarray Manufacturing

Microarray Chip Fabrication

• Glass Slides• Nitrocellulose Membranes• Silicone Slides

Protein

Immobilization

• Diffusion• Affinity Binding• Adsorption• Covalent cross linking

Microarray Signa

l Detection

• Direct Labelling• Sandwich immunoassay

Protein Microarray-Applications• Diagnostics:

– involves the detection of antigens and antibodies in blood samples

– profiling of sera to discover new disease biomarkers

– monitoring of disease states and responses to therapy in personalized medicine

• Proteomics: protein expression profiling i.e. which proteins are expressed in the lysate of a particular cell.

• Protein functional analysis: identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, enzymatic substrates and receptor ligands.

• Treatment: development of antigen-specific therapies for autoimmunity, cancer and allergies; the identification of small molecule targets that could potentially be used as new drugs.

Antibody Microarray

• Antibody microarray (also known as antibody array) is a specific form of protein microarray, a collection of capture antibodies are spotted and fixed on a solid surface such as glass, plastic or silicon chip, for the purpose of detecting antigens.

• Invented by Tse Wen Chang in 1983 and further developed by Roger Ekins and colleagues.

• ELISA is a form of Antibody Micro assay this is extensively used in Diagnostics.

Antibody Microarray-Methodology

• Similar technique as Protein microarray, a solid support such as silicon slide or glass slide is imbedded with a specific antibody or a specific antigen.

• The membrane or support is blocked for non-specific binding by BSA or other blockers.

• The cell lysate or the sample with protein is applied to the microarray and incubated for 15 mins.

• After incubation, the excess sample is drained off by washing in Tris-Buffer solution.

• Antibodies labelled with fluorescent dye against the target antigen/ protein is added and incubated.

• Additional antibodies is washed off and microassayed under laser or appropriate detection technique.

Antibody Microarray-Methodology

• Fig a: Direct labelling, single-capture antibody experiments. All proteins in a sample are labelled (red haloes), thereby providing a means for detecting bound proteins following incubation on an antibody microarray.

• Fig b: Dual-antibody (capture and read-out antibody) sandwich immunoassays. Proteins captured on an antibody microarray are detected by a cocktail of tagged detection antibodies, which are matched to the spotted antibodies. The detector antibody tag is then measured by binding of a labelled read-out antibody.

Antibody Microarray-Application

• Diagnostics: ELISA, a form developed for HIV and other viral detection is extensively used. Most of the confirmation tests for other infections and diseases are carried out by Antibody microarray.

• HLA compatibility and related studies.• Profiling studies on serum/ blood samples or tissue lysates.• Cancer studies: For biomarker identification and identification of

new biomarkers.

Conclusion

Microarray Technique Used For Advantage

DNA Microarray DNA expression and identification

Cheap and can be used for mass diagnostics

SAGE DNA expression and identification

High Throughput

Protein Microarray Protein Identification in cell lysates

High Throughput

Antibody Microarray Biomolecule identifications and expressions

Cheap and can be used for mass diagnostics

Cell Microarray Cell surface biomolecules expression & identification

Cellular studies, MHC studies, Biomarker development

Tissue Microarray Tissue engineering and Histology studies

Cheap and fast Tissue histology studies

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Seminar on

MultidimensionalLiquid chromatography

Presented byGourab Ray

Under the guidance of

Dr. T. Ravi KiranAssistant professor

Dept. of Microbiology and BiotechnologyBangalore University

Bangalore-56

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The vast interest in proteins has led to a significant and persistent effort in the development of analytical strategies for proteome analysis.

Multidimensional liquid chromatography (MDLC) allows separation of complex mixtures by using multiple columns with different stationary phases (Giddings et al., 1984).

These columns are coupled orthogonally (90° to each other) which means that fractions from the first column can be selectively transferred to other columns for additional separation. This enables separation of complex mixtures that cannot be separated using a single column.

Mass spectrometry provides more information than PDA and is often the detector of choice in MDLC.

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MDLC

MS

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Separation

Gel Electrophoresis (1D and 2D) MDLC

Tryptic Digest

Mass Spectrometric Analysis Mass Spectrometric Analysis

Database Search

Protein Mixture (standard proteins or cell lysate)

Tryptic Digest

MDLC

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1. Two HPLC columns working in parallel receive alternating elutes from a bank of six size exclusion columns in series.

2. After sample injection and separation by size exclusion chromatography, elute from the size exclusion columns is directed to HPLC column 1 using a four-port valve (thick line).

3. While the peptides are trapped in this column, HPLC column 2 is eluted and the sample is directed to the detector and fraction collector (broken line).

4. After flushing and equilibrating column 2, the valves are reversed allowing column 2 to be loaded with the next fraction from the size exclusion separation, while column 1 is eluted.

Continuous multidimensional

chromatography with column switching

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Mass spectrometry (MS):

The successful combination of MDLC separations with MS for protein and peptide analysis was achieved with the advent of the soft ionization techniques MALDI and ESI. 

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Types of MDLC:A. Off-line MDLC: based on fractions collection in the first dimension and their analysis

in the following dimensionB. On-line MDLC: involves a direct transfer of the eluent from the first dimension onto

the next one, with no flow interruption

A) In an off-line setup the sample is first separated by SCX and fractions are collected. The fractions can be processed if needed and are subsequently separated by RP-LC and analyzed by MS.

B) An example of an on-line column switching setup. The sample is first loaded onto the SCX column and eluted stepwise onto the trap column. The sample is then desalted and subsequently eluted onto the analytical RP column followed by MS analysis.

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MDLC is usually applied for analytes present at very low concentrations in

complex samples.

Thus, a first dimension column with sufficient sample capacity is required for

accommodating sufficient injection volumes for trace component

determination.

Size of the first dimension column: 2-4mm ID/capillary/microflow/nanoflow

columns.

Injection volume: 10-1000µL.

As a rule of thumb, the second dimension columns should allow fast

separation in order to have optimal fractionation rate in the first dimension.

Size of the second column: column with diameter 10µm.

Size of the column:

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•Reversed phase (RP)

•Ion exchange chromatography (IEC)

•Size exclusion chromatography (SEC)

•Cation exchange chromatography (CX)

•Anion exchange chromatography (AX)

•Normal phase chromatography (NP)

Depending on the analytes and type of sample. All combinations provide high selectivity as well as peak capacity compared to 1D LC.

Schematic illustration of interactions between polar, apolar, negatively charged sites of a tryptic peptide and different stationary phases

Combination of different separation mechanisms in MD LCColumns with different separation mechanisms should be selected to achieve the needed separation orthogonality in MD LC.

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Reversed phase (RP)

Includes any chromatographic method that uses a hydrophobic stationary phase.

Salts and the majority of components used in digestion protocols tend to remain in the low

organic solvent .

The use of RP columns in both dimensions can be achieved either with stationary phases

showing different selectivity operated with the same mobile phase or with the same

stationary phase but changing pH of the mobile phases in the two dimensions.

Utilizing a RP-RP system has several advantages including high peak capacity in the first

separation dimension.

This permits the collection of multiple fractions with minimal content overlap.

In addition, no peptide losses were observed in the first RP dimension and the mobile

phases were salt free and compatible with MS detection.

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Ionexchange chromatography (IEC)

Ion exchange columns are used to separate ions and molecules that can be easily

ionized. Separation of the ions depends on the ion's affinity for the stationary phase,

which creates an ion exchange system.

The electrostatic interactions between the analytes, moble phase, and the stationary

phase, contribute to the separation of ions in the sample.

Only positively or negatively charged complexes can interact with their respective

cation or anion exchangers.

Common packing materials for ion exchange columns are amines, sulfonic acid,

diatomaceous earth, styrene-divinylbenzene, and cross-linked polystyrene resins.

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Size Exclusion Chromatographic Columns

Size Exclusion Chromatographic columns separates molecules based upon

their size, not molecular weight.

A common packing material for these columns is molecular sieves. Zeolites are

a common molecular sieve that is used.

The molecular sieves have pores that small molecules can go into, but large

molecules cannot. This allows the larger molecules to pass through the column

faster than the smaller ones.

Other packing materials for size exclusion chromatographic columns are

polysaccharides and other polymers, and silica.

The pore size for size exclusion separations varies between 4 and 200 nm. 

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Common applications:

MD LC is widely used for separation in proteomics as well as in industrial applications.

Proteins- SCX-RP column• Gao et al. used this method separate 53 proteins from human liver tissue• Degradation products (apolipoprotein) from E.Coli and human plasma were

identified.

Peptides- SCX-RP or AX-RP column• Peptides, either as digested proteins in proteomics or endogenous as in

peptidomics are frequently separated by MDLC.• More than 1800 phosphopeptides were identified in HeLa cells.

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Advantages: Basic proteins and membrane proteins can be separated easily Proteins separated in the liquid phase do not need to be stained in order to be detected Important fact that LC methods can separate peptides as well as proteins Ability to couple LC columns directly to the MS Entire analytical process from sample preparation to peptide mass profiling can be automated

Disadvantages: Visual aspects of protein separation by 2D-PAGE are lost, including the PI and molecular mass data from the positions of spots on the gel (these data can be used in database searches)Peak drift with column ageing in all forms of partitioning based separations has been identified as the major issue.

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Conclusion:

MD LC will be more frequently utilized in the future, due to the increasing need for automatic, high throughput comprehensive and target analysis of complex samples.

At present, miniaturized on-line MD LC systems are becoming more common with capillary/microflow and nanoflow columns due to the increasing need of detecting different components at very low concentration levels and such systems will be more frequently applied also in routine analyses.

In conclusion, many different methods for protein separation have been described in the last 30 years. Furthermore, all these separations are dramatically evolving. The question remains which approach is most suitable for an experiment.

The choice is dependent on the analytical question, the available equipment, the amount of sample and analysis time available and the experience of the operator.

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