protein microarray-types and approaches.pptx

Protein Microarray
A high -throughput screening method

What is microarray ?
 Arrays are solid support upon which a
collection of gene specific probe have
been placed at defined locations, either
by spotting or, direct synthesis.
 The term ‘micro” refers to the small
size of the solid support.
 There are 2 type of Arrays
1 2 3 4
5 6 7 8
9 10 11 12
13 14 15 16
 DNA micro-Array (An indirect method for proteomic)
 Protein micro-Array (A direct method for proteomic)

INTRODUCTION
• Protein microarrays are a high-throughput technology used to
analyze the expression levels and functional activity of thousands
of proteins simultaneously.
• Protein microarrays consist of a solid support (such as a glass
slide or a membrane) that has been coated with thousands of
different proteins in a defined pattern or array.
• Each protein spot on the array represents a different protein, and
multiple replicates of each protein are typically included on the
array to ensure data reproducibility
3

Comparison with other methods (e.g., ELISA, Western
blotting)
Here are some of the key differences between these methods:
Throughput: Protein microarrays allow for the simultaneous screening of hundreds or
thousands of proteins in a single experiment, while ELISA and Western blotting typically
only allow for the analysis of a few proteins at a time.
Sensitivity: Protein microarrays can detect low abundance proteins with high
sensitivity, while ELISA and Western blotting may have limited sensitivity for low
abundance proteins.
Specificity: Protein microarrays can offer higher specificity by allowing for the
screening of multiple proteins in a single experiment, while ELISA and Western blotting
may have limitations in terms of cross-reactivity and specificity.
Sample requirements: Protein microarrays require only small amounts of sample,
while ELISA and Western blotting typically require larger amounts of sample.
Cost: Protein microarrays can be cost-effective due to the high throughput and low
sample requirements, while ELISA and Western blotting can be more expensive due to
the need for multiple experiments or larger sample volumes.
4

 A protein microarray (or protein chip) is a 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 lies in the fact that large numbers of
proteins can be tracked in parallel
Motivation for development-
 Developed due to the limitations of using DNA microarrays for
determining gene expression levels in proteomics.
 Additionally post-translational modifications, which are often
critical for determining protein function, are not visible on DNA
microarrays.

Protein chip consists of a support surface such as
glass slide, nitrocellulose ,bead or micro-titer
plate to which array of capture proteins is bound.
Probe molecules typically labelled with a
fluorescent dye are added to the array.
A reaction between the probe and the immobilized
protein emits a fluorescent signal and that is read
by a laser scanner
Principle-

Solid Supporting Material
• The most common supporting materials in use includes Aldehyde, carboxylic ester,
nitrocellulose membrane, polystyrene, Agarose/polyacrylamide gel, hydrogel.
• An ideal surface for protein microarray fabrication has to be capable of
I. Immobilizing proteins
II. Preserving three-dimensional (3-D conformation of protein
III. Should not change the chemical nature of the protein
8

The probe on chip
A variety of material can be immobilize on the protein chip based on the
specific requirement .This include
1.Antibodies
2.Antigens
3.Aptamers(nucleic acid based ligand)
4.Affibodies(small ,robust proteins engineered to bind to a large number
of target proteins or, peptides with high affinity , imitating monoclonal
antibodies , and are therefore a member of the family of antibody
mimetics.)
5.Full length protein or , their domain

The sources of probes on chip-
1. Cell lysates-if you are interested in full length
protein) by using different chromatographic
techniques like affinity chromatography, ion
exchange chromatography).
2. Recombinant DNA Technology-

3.Synthetic peptides- The established method for the production of synthetic
peptides in the lab is known as solid-phase peptide synthesis (SPPS).

4. Cell free translation system-
Cell-free protein synthesis (CFPS) ,also known as in vitro protein
synthesis ,is the production of protein using biological machinery
in a cell free system ,that is, without the use of living cells.
Common components of a cell-free reaction include a
1. Cell extract
2. An energy source( phosphoenol pyruvate ,acetyl
phosphate,and creatine phosphate)
3. Supply of amino acids
4. Cofactors ,such as mg2+(for keeping ribosome intact)
5. The DNA with the desired gene.
6. The necessary cell machinery including ribosomes ,
aminoacyl-trna synthetases ,translation initiation and
elongation factor, etc

Fabrication of protein microarray
Protein microarrays are typically prepared by immobilizing
proteins onto a microscope slide using a standard contact
spotter or non contact microarray.
PROTEIN PRODUCTION
Recombinant antibodies(phage display)
•Using antibody-fragment encoding genes (VH and VL) and
bacteriophage capsid gene fusion, this technology enables sets of
human antibody libraries to be stored in prokaryotic systems where they
can be readily expressed by phage infection.
• Prokaryotic expression systems promise large-scale antibody
production in short time periods. In addition, this system generates
antibody fragments lacking the Fc domain rather than intact IgG,
eliminating nonspecific binding to the Fc receptor.
•The recombinant antibodies are expressed and displayed in the phage
capsid, and then purified using column chromatography.

• Recently, other methods have been developed to generate recombinant
antibodies in eukaryotic expression systems (i.e., yeast display) or even in
vitro environments (i.e., mRNA display, ribosome display), which provide
additional advantages for recombinant antibody fabrication.
•Fabrication of functional protein microarrays faces an even bigger
challenge due to the need for large amounts of highly purified proteins.
•. To overcome these hurdles, high-throughput protein purification protocols
have been developed using both Saccharomyces cerevisae (yeast) and E.
coli protein expression systems.
• Using a batch purification protocol in a 96-well format, >4000
recombinant proteins can be overexpressed and purified in yeast or E. coli
(Jeong et al., 2012).
•Because it is challenging for most labs to purify large numbers of proteins,
Angenendt et al. (2006) developed an alternative technology in protein
chip fabrication, dubbed nucleic acid- programmable protein array
(NAPPA).

• Spotting plasmid DNAs with capture antibodies allows for the generation
of a protein microarray via simultaneous in situ transcription/translation
reactions and protein immobilization on the printed slides.
•A significant benefit of this approach is that the printed template DNA
microarray can be stored for a long time, and the resulting protein
microarrays are always freshly made.
•This method allows the generation of up to 13,000 protein spots on one
slide without laborious cloning and expression vectors.
• However, such arrays have not flourished due to low protein yield and
difficulties in producing large proteins (e.g., >60 kD).
• For reverse-phase protein microarrays proper samples should be
isolated from cell culture; frozen, ethanol-fixed, or paraffin-embedded
tissue or laser captured microdissections of cell populations from certain
tissues (Charboneau et al., 2002; Espina et al., 2007)

•A variety of slide surfaces can be used. Popular types include aldehyde-
and epoxy-derivatized glass surfaces for random attachment through
amines , nitrocellulose , or gel-coated slides and nickel-coated slides
for afﬁnity attachment of His6-tagged proteins.

Nucleic Acid programmable protein
Array(NAPPA)-
Avidin is a tetrameric biotin binding protein. Biotin is a water soluble vitamin.

Drawbacks of NAPPA-
The main drawback of this method is the extra and tedious preparation steps
at the beginning of the process
Resulting protein array is not pure because the proteins are co-localized
with their DNA template and capture antibodies.
Protein in situ array(PISA)
Unlike , NAPPA ,PISA completely bypasses DNA immobilization as the
DNA template is added as a free molecule in the , reaction mixture ,so in
the end the DNA will not be present in the well of the chip.

Types of protein microarrays
There are several types of protein microarrays, each
with its own strengths and limitations. Here are some
of the most common types
Protein
microarray
Analytical
Functional
Reverse phase
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Analytical microarrays
• Analytical microarrays are typically used to profile a
complex mixture of proteins in order to measure binding
affinities and protein expression levels of the proteins in
the mixture.
• In this technique, a library of antibodies, aptamers, or
affibodies is arrayed on a glass microscope slide.
• The array is then probed with a protein solution.
• Antibody microarrays are the most common analytical
microarray .
• These types of microarrays can be used to monitor
differential expression profiles and for clinical diagnostics.
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Functional protein microarrays
• Functional protein microarrays is different from
analytical arrays. Functional protein arrays are
composed of arrays containing full-length functional
proteins or protein domains.
• These protein chips are used to study the
biochemical activities of an entire proteome in a
single experiment.
• They are used to study numerous protein
interactions, such as protein-protein, protein-DNA,
and protein-RNA interactions.
• Functional protein microarrays can be used to study
a wide range of biological processes, including
signal transduction, transcriptional regulation, and
apoptosis.
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Reverse-phase protein microarrays :
• Reverse-phase protein microarrays : reverse phase
protein microarray (RPA). In RPA, cells are isolated
from various tissues of interest and are lysed.
• The lysate is arrayed onto a nitrocellulose slide using a
contact pin microarrayer.
• The slides are then probed with antibodies against the
target protein of interest, and the antibodies are
typically detected with chemiluminescent, fluorescent,
or colorimetric assays.
• Reverse-phase protein microarrays can be used for
biomarker discovery, drug target validation, and the
study of protein expression patterns in disease.
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Reverse-Phase Protein Microarray
•Involves complex samples, such as tissue lysates.cells are isolated from
various tissues of interest and lysed.The lysate is arranged onto the
microarray & probed with antibodies against the target protein of
interest.These antibodies are typically detected with
chemiluminescent,fluoresent or colorimetric assays.
• This type of microarrays was first established by Paweletz and
colleagues to monitor histological changes in prostate cancer patients.
Using this method, they successfully detected microscopic transition
stages of pro-survival checkpoint protein in three different stages of
prostate cancer: normal prostate epithelium, prostate intraepithelial
neoplasia, and invasive prostate cancer.
•The high degree of sensitivity, precision and linearity achieved by reverse-
phase protein microarrays enabled this method to quantify the
phosphorylation status of some proteins (such as Akt and ERK) in these
samples; phosphorylation was statistically correlated with prostate cancer
progression.

protein microarray-types and approaches.pptx

Types of protein Microarray-
• Analytical microarrays are also known as capture arrays,uses a
library of antibodies , Aptamers or Affibodies arrayed on the
support surface. These are used as capture molecules since each
binds specifically to a particular protein .
• The array is probed with a complex protein solution such as a
cell lysate. This type of microarray is especially useful in
comparing protein expression in different solutions.
• For instance the response of the cells to a particular factor can be
identified by comparing the lysates of cells treated with specific
substances or grown under certain conditions with the lysates of
control cells. Another application is in the identification and
profiling of diseased tissues.

Analytical Protein Microarray
• The first model to demonstrate the application of antibody arrays
was the “analyte-labeled” assay format. In this format, proteins are
detected after antibody capture using direct protein labeling .
•Some limitations have to be considered because this method lacks
specificity in protein target labeling and has poor sensitivity for low
abundance proteins. Moreover, targeted protein labeling may lead
to the epitope destruction due to some chemical reaction.

LIMITATIONS
•Antibodies are the most popular protein capture reagents, although their
affinity and/or specificity can vary dramatically .
• Many antibodies may cross-react with proteins other than their expected
target proteins when tested on functional protein microarrays, especially
when multiple analyte detection is employed.
•The need for highly specific antibodies has become a major challenge in
analytical protein microarrays because nonspecific binding will lead to
large numbers of false positive result.
•Another challenge comes from producing a large number of antibodies in
a high-throughput fashion. Recombinant antibodies have become a
promising means to overcome this problem; however, they are not ready
for prime time performance yet.

•Another model of antibody array provides higher sensitivity using
the “sandwich” assay format. This format employs two different
antibodies to detect the targeted protein .
• One antibody, called the capture antibody, immobilizes the
targeted protein on the solid phase, while the other antibody,
called the reporter or detection antibody, generates a signal for
the detection system. Using two antibodies significantly increases
the specificity and sensitivity of the “sandwich” assay format, even
at femtomolar levels .
•These assays offer a multiplexed format of the original Enzyme-
linked Immunosorbent Assay (ELISA).

•Analytical microarrays(or antibody microarrays) have
antibodies arrays on solid surface,and are used to detect
proteins in biological samples.
•Often a second is used to detect a protein that is captured by
the antibody attached to the solid phase,in a principle similar
to that of sandwich immunoassay, in which the first antibody
is spotted on the array and then a captured antigen on the
chip is detected with a second antibody that recognises a
different part of antigen.
•Analytical protein arrays can be used to monitor protein
expression levels or for biomarker identiﬁcation, clinical
diagnosis, or environmental/ food safety analysis.

Functional microarray-
• also known as( target protein arrays ) are constructed by
immobilizing large numbers of purified proteins and are used to
identify protein-protein , protein-DNA ,protein-RNA and protein
small molecule interaction , to assay enzymatic activity and to
detect antibodies and demonstrate their specificity .
• They differ from analytical arrays in that functional protein
arrays are composed of arrays containing full length functional
or protein domains .
• These protein chips are used to study the biochemical activities
of the entire proteome in a single experiment.

Functional Protein Microarray
•Also known as target protein array.With functional protein
microarrays purified recombinant protein are immobilised onto
the solid phase.
•Functional protein microarrays have recently been applied to
many aspects of discovery based biology,including protein-
protein,protein- lipid,protein-DNA,protein-drug,&protein –
peptide iinteractions.
•These can be used to identify enzyme substrates.

•These can also be used to detect antibodies in a biological
specimen to profile an immune response.
•The first use of functional protein microarrays was
demonstrated by Zhu et al. (2001) to determine the
substrate specificity of protein kinases in yeast.
• Protein microarrays enable us to study many post-
translational modifications (i.e., phosphorylation, acetylation,
ubiquitylation, S- nitrosylation) in a large-scale fashion,
which is critical for understanding cellular protein synthesis
and function.

Application of protein
Microarray-
• Functional protein microarrays provide a flexible platform that can
characterize a wide range of biochemical properties of spotted
proteins.
• To date,these assay have been successfully developed to detect
various types of protein-binding properties ,such as protein-protein,
protein-DNA , protein-RNA , protein-drug and identify substrate of
various classes of enzymes ,such as protein kinase , ligases.
• Biomarkers are a crucial tool in the expeditious detection of
infection or diagnosis of autoimmune disease.
• Protein post –translational modification are one of the most
important mechanisms to directly regulate protein activity . Among
the hundreds of PTMS identified ,in addition to glycosylation,
enzyme-dependent , reversible protein de(phosphorylation) and
de(acetylation) are perhaps the most well studied.

• A new trend in the field of host –pathogen
interactions is to use host protein microarrays to
survey relationship between a pathogenic factor
of interest and the host proteome.this idea is
particularly suited for studying host-virus
interaction because ,after entering the host cells
,the viral genome and proteins are in direct
physical contact with the host compoments
,where by a pathogen can hajack/exploit the host
pathways and machineries for its own replication.

Challenges-
Despite the considerable investments made by several companies ,
protein chip has yet to flood the market .Manufacturers have found that
the proteins are actually quite difficult to handle . A protein chip
requires a lot more steps in its creation than does a DNA chip.
Challenges include
Finding a surface and a method of attachment that allows the proteins
to maintain their secondary/tertiary structure and thus their biological
activity and their interaction with other molecules.
Producing an array with a long shelf life so that proteins on the chip do
not denature over a short time.

• Identifying and isolating antibodies or other capture molecules
against every protein in the human genome.
• Quantifying the levels of bound protein while assuring sensitivity
and avoiding background noise.
• Extracting the detected protein from the chip in order to further
analyse it.
• Reducing non-specific binding by the capture agents.
• The capacity of the chip must be sufficient to allow as complete a
representation of the proteome to be visualized as possible ; abudant
protein overwhelm the detection of less abundant proteins such as
signalling molecules and receptors ,which are generally of more
therapeutic interest.

Case studies/examples
Examples of successful drug discovery using protein microarrays
• Discovery of a selective inhibitor of Bromodomain-containing protein
4
• Identification of a biomarker for Alzheimer's disease
• over 900 human proteins to identify autoantibodies in the serum of
patients with autoimmune disease.
42

Discovery of a selective inhibitor of BRD4:
• Bromodomain-containing protein 4 (BRD4) is a promising target for cancer therapy,
but developing selective inhibitors has been challenging due to the high structural
similarity of BRD4 with other bromodomain-containing proteins.
• Researchers used a protein microarray containing 42 human bromodomain proteins
to screen for inhibitors that selectively bind to BRD4.
• They identified a compound that binds selectively to BRD4 and showed potent
antiproliferative activity against multiple cancer cell lines. The compound has since
been further optimized and is being developed as a potential cancer therapy
43

Identification of a biomarker for Alzheimer's
disease:
• Researchers used a protein microarray containing over 9,000 proteins to screen for
proteins that are differentially expressed in the brains of Alzheimer's disease patients
compared to healthy controls.
• They identified a protein called REST, which is a transcriptional repressor that
regulates neuronal gene expression.
• REST was found to be significantly decreased in the brains of Alzheimer's disease
patients, and further studies showed that it may be a potential biomarker for the
disease.
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Development of a diagnostic test for autoimmune
disease:
• Researchers used a protein microarray containing over 900 human proteins to
identify autoantibodies in the serum of patients with autoimmune disease.
• They identified a panel of 11 autoantibodies that are highly specific for
autoimmune disease, and developed a diagnostic test based on these
autoantibodies.
• The test has been shown to have high sensitivity and specificity for autoimmune
disease, and is being developed for clinical use .
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APPLICATIONS:-
There are five major areas where protein arrays are being
applied:diagnostics,proteomics,protein functional analysis,antibody
characterisation & treatment.
Diagnostics involves the detection of antigens & antibodies in
blood samples;to discover new disease biomarkers;the monitoring
of disease states & responses to therapy in personalised
medicine;the monitoring of environment & food.
Proteomics pertains to protein expression profilling i.e;which
proteins are expressed in the lysate of a part of cell

 Protein functional analysis is the identification of protein-protein
interactions,protein- phospholipid interactions,small molecule
targets,enzymatic substrates & receptor ligands.
 Antibody characterization is characterizing cross
reactivity,specificity & mapping epitopes.
 Treatment development involves the development of antigen-
specific therapies for autoimunity,cancer & allergies;the
identification of small molecule targets that could potentially be
used as new drugs.

Applications in Basic Research
•Development of new assays
An obvious advantage of functional protein
microarrays is their ability to provide a flexible platform that can
characterize a wide range of biochemical properties of spotted
proteins.
To date, these assays have been successfully
developed to detect various types of protein binding properties,
such as protein-protein, protein-DNA, protein-RNA, protein-lipid,
protein-drug, and protein-glycan interactions.
And identify substrates of various classes of
enzymes, such as protein kinases, ubiquitin/SUMO E3 ligases, and
acetyltransferases, to name a few.

Detection of Protein-Binding Properties
Protein-protein interaction
•Zhu and Snyder (2001) developed the first proteome microarray
composed of ~5800 recombinant yeast proteins (>85% of the yeast
proteome) and identified binding partners of calmodulin and
phosphatidylinositides (PIPs).
•They first incubated the microarrays with biotinylated bovine
calmodulin and discovered 39 new calmodulin binding partners.
•In addition, using liposomes as a carrier for various PIPs, they
identified more than 150 binding proteins, >50% of which were
known membrane-associated proteins.

Protein-Peptide Interaction—
•MacBeath and colleagues fabricated protein domain microarrays to
investigate protein-peptide interactions that might play an important
role in signaling in a semi-quantitative fashion.
•They constructed an array by printing 159 human Src homology 2
(SH2) and phosphotyrosine binding (PTB) domains on the aldehyde-
modified glass substrates and incubated the arrays with 61 peptides
representing tyrosine phosphorylation sites on the four ErbB
receptors.
•Eight concentrations of each peptide (10 nM to 5 mM) were tested in
the assay, allowing semi-quantitative measurement of the binding
affinity of each peptide to its protein ligand

Protein-DNA Interaction—
•Protein microarrays have also been applied extensively and
successfully to characterize protein-DNA interactions (PDIs).
•In an earlier study, Snyder and colleagues screened for novel DNA-
binding proteins by probing yeast proteome microarrays with
fluorescently labeled yeast genomic DNA .
• Of the ~200 positive proteins, half were not previously known to bind
to DNA. By focusing on a single yeast gene, ARG5,6 , encoding two
enzymes involved in arginine biosynthesis, they discovered that its
protein product bound to a specific DNA motif and associated with
specific nuclear and mitochondrial loci in vivo .

Protein-Small Molecule Interaction—
•Discovering new drug molecules and drug targets is another field in which
protein microarrays have shown its potential.
•For example, Huang et al. (2004) incubated biotinylated small-molecule
inhibitors of rapamycin (SMIRs) on the yeast proteome microarrays, and
obtained the binding profiles of the SMIRs across the entire yeast
proteome.
• They identified candidate target proteins of the SMIRs, including Tep1p, a
homolog of the mammalian PTEN tumor suppressor, and Ybr077cp
(Nir1p), a protein of previously unknown function, both of which are
validated to associate with PI(3,4)P2, suggesting a novel mechanism by
which phosphatidylinositides might modulate the target-of-rapamycin
pathway

Protein-Glycan Interaction—
•Protein glycosylation, a general posttranslational modification of proteins
involved in cell membrane formation, dictates the proper conformation of
many membrane proteins, retains stability on some secreted
glycoproteins, and plays a role in cell-cell adhesion.
Profiling monoclonal antibody specificity—
•Antibodies are widely applied for many purposes in proteomic studies. Because
of their specificity, monoclonal antibodies (MAb) are a better option compared to
polyclonal antibodies for most applications.
•Jeong et al. (2012) combined immunization with live human cells and
microarray-based analysis to develop a rapid identification method of
monospecific monoclonal antibody (mMAb).
• Because a human proteome microarray composed of ~17,000 individually
purified full-length human proteins was used in the monoclonal antibody binding
assays, antibodies that only recognized a single antigen on the microarrays
could be identified as highly specific mMAbs

Protein posttranslational modification
Protein phosphorylation—
•Protein phosphorylation plays a central role in almost all aspects of
cellular processes.
•The application of protein microarray technology to protein
phosphorylation was first demonstrated by Zhu et al. (2000). They
immobilized 17 different substrates on a nanowell protein microarray,
followed by individual kinase assays with almost all of the yeast
kinases (119/122).
•This approach allowed them to determine the substrate specificity of
the yeast kinome and identify new tyrosine phosphorylation activity.

Protein Ubiquitylation—
•Ubiquitylation is one of the most prevalent PTMs and controls almost all
types of cellular events in eukaryotes.
•To establish a protein microarray-based approach for identification of
ubiquitin E3 ligase substrates, Lu et al. (2008) developed an assay for
yeast proteome microarrays that uses a HECT-domain E3 ligase, Rsp5, in
combination with the E1 and E2 enzymes.
•More than 90 new substrates were identified, eight of which were
validated as in vivo substrates of Rsp5. Further in vivo characterization of
two substrates, Sla1 and Rnr2, demonstrated that Rsp5-dependent
ubiquitylation affects either posttranslational processing of the substrate or
subcellular localization.

Protein Acetylation—
•Histone acetylation and deacetylation, which are catalyzed by histone
acetyltransferases (HATs) and histone deacetylases (HDACs),
respectively, are emerging as critical regulators of chromatin structure and
transcription. It has been hypothesized that many HATs and HDACs might
also modify non-histone substrates.
• For example, the core enzyme, Esa1, of the essential nucleosome
acetyltransferase of H4 (NuA4) complex, is the only essential HAT in yeast,
which strongly suggested that it may target additional non-histone proteins
that are crucial for cell to survive.
•To identify non- histone substrates of the NuA4 complex, Lin et al. (2009)
established and performed acetylation reactions on yeast proteome
microarrays using the NuA4 complex in the presence of [14C]-Acetyl-CoA
as a donor.
• Surprisingly, 91 proteins were found to be readily acetylated by the NuA4
complex on the array

S-Nitrosylation—
•S-nitrosylation is independent of enzyme catalysis but an important PTM
that affects a wide range of proteins involved in many cellular processes.
• Recently, Foster et al. (2009) developed a protein microarray-based
approach to detect proteins reactive to S-nitrosothiol (SNO), the donor of
NO+ in S-nitrosylation, and to investigate determinants of S-nitrosylation
(Foster et al., 2009).
•S-nitrosocysteine (CysNO), a highly reactive SNO, was added to a yeast
proteome microarray, and the nitrosylated proteins were then detected
using a modified biotin switch technique.
• The top 300 proteins with the highest relative signal intensity were
further analyzed, and the results revealed that proteins with active-site
Cys thiols residing at N-termini of alpha-helices or within catalytic loops
were particularly prominent

Applications in Clinical Research
Extending the applications in basic research, protein
microarrays have proven highly useful in clinical research because the
development of almost all diseases is related to protein function and interaction.
In addition, the protein microarray format can be directly employed to develop
highly sensitive and specific diagnostic and detection tools
Host-microbe interactions
•A new trend in the field of host-pathogen interactions is to use host
protein microarrays to survey relationships between a pathogenic factor of
interest and the host proteome.
•This idea is particularly suited for studying host-virus interactions
because, after entering the host cells, the viral genome and proteins are in
direct physical contact with the host components, whereby a pathogen
can hijack/exploit the host pathways and machineries for its own
replication. This approach would alleviate the problems associated with
RNAi-based screening in identifying direct host target

•Pathogen entry and infection in the host cell are a series of processes
that abuse protein pathways and interactions.
•Li et al. (2011) demonstrated the use of protein microarrays to study
conserved serine/threonine kinase of herpesvirus that play an important
role in their replication in human cells.
• They identified shared substrates of the conserved kinases from herpes
simplex virus, human cytomegalovirus, EBV, and Kaposi’s sarcoma-
associated herpesvirus using human proteomic chips.
• From this study, they found that the histone acetyltransferase TIP60, an
upstream regulator of the DNA damage response pathway, was essential
for herpesvirus replication.
•This finding is promising for broad-spectrum anti-viral development.

Biomarker identification
•Biomarkers are a crucial tool in expeditious detection of infection or
diagnosis of autoimmune diseases. In most cases, production of
antibodies against pathogens or autoantibodies in blood serum is
correlated with infection or occurrence of autoimmune diseases.
•Therefore, to find a powerful biomarker, protein microarrays can be used
to directly detect antibodies that statistically correlate with the
corresponding disease in a patient’s serum.
•Zhu et al. (2006) developed the first viral protein microarray to detect
biomarkers for severe acute respiratory syndrome (SARS).

•This array, which consisted of all SARS coronavirus (SARS-CoV)
proteins, as well as proteins of five additional coronaviruses, could readily
distinguish serum samples as SARS-positive or SARS-negative with 94%
accuracy compared to the traditional ELISA method.
•More recently, human protein microarrays have been widely used to
identify biomarkers for a variety of autoimmune diseases
•In additions, there have been a series of studies that employed pathogen
protein microarrays to profile serological responses following infection. For
examples, protein microarrays have been developed in bacteria and
viruses for biomarker identification in various infectious diseases

Future Prospects
Protein microarrays have become one of the most powerful tools in
proteomic studies and can be applied with many different purposes.
High-throughput processing has become the trend due to cost reduction
and high productivity of results. Given a high-throughput and parallel
system, protein microarrays will speed up new findings in protein
interactions for basic research as well as clinical research purposes.
Reduction of sample volume usage is one important factor that
demonstrates the superiority of this technology compared to other
techniques.
This factor is especially important for clinical research that uses precious
samples, such as human serum samples. In addition, the high sensitivity
and specificity of protein microarrays provides a powerful tool in
quantifying and profiling proteins.

•Despite many successful applications of protein microarrays,
limitations of this technology still leave many challenges to be
overcome.
•The large-scale production of high-quality antibodies using
recombinant platforms is still hard to be applied due to the
complexity of expression and purification procedures.
•Another consideration is to perform high-throughput detection
without sample labeling. Label-free detection systems should be
the future of protein microarrays.
• In conclusion, although this technique still needs to be explored,
it would not be surprising if after several years, this technique is
the leading technology in proteomic and diagnostic fields.

Reference
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protein microarray-types and approaches.pptx

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protein microarray-types and approaches.pptx