Multiplex tools- Microarray techniques / Microassay techiniques and Multidimentional Liquid Chromatography

1. Multiplex Analysis
By: Gourab Ray
Research Scholar
Dept. of Biotechnology
Bangalore UniversityUnder Guidance of:
Dr. Ravikiran T.
Professor, Dept. of Biotechnology,
Bangalore University

2. Agenda
• Multiplex Analysis & Types of Multiplex assays.
– Protein Microarray
– Antibody Microarray
• Multidimensional Liquid Chromatography
– Principle
– Types
– Applications

3. 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.

4. 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

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. Protein Microarray-Microarray
Manufacturing
Microarray
Chip
Fabrication
• Glass Slides
• Nitrocellulose Membranes
• Silicone Slides
Protein
Immobilizatio
n
• Diffusion
• Affinity Binding
• Adsorption
• Covalent cross linking
Microarray
Signal
Detection
• Direct Labelling
• Sandwich immunoassay

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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

17. Seminar on
Multidimensional
Liquid chromatography
Presented by
Gourab Ray
Under the guidance of
Dr. T. Ravi Kiran
Assistant professor
Dept. of Microbiology and Biotechnology
Bangalore University
Bangalore-56
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18. 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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19. 6/9/2015 34
MDLC
MS

20. 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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21. 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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22. 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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23. Types of MDLC:
A. Off-line MDLC: based on fractions collection in the first dimension and their analysis
in the following dimension
B. 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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24. 6/9/2015 40
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:

25. •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 LC
Columns with different separation mechanisms
should
be selected to achieve the needed separation
orthogonality in MD LC.
6/9/2015 41

26. 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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27. Ionexchange chromatography (IEC)
6/9/2015 43
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.

28. 6/9/2015 44
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.

29. 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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30. 6/9/2015 49
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.

31. 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.
6/9/2015 50

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