Protein Chip

Seminar Report 2015

Chapter 1
INTRODUCTION
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.[1] Its main advantage lies in the fact that large
numbers of proteins can be tracked in parallel. The chip consists of a support surface
such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an
array of capture proteins is bound.[2] Probe molecules, typically labeled with a
fluorescent dye, are added to the array. Any reaction between the probe and the
immobilised protein emits a fluorescent signal that is read by a laser scanner.[3] Protein
microarrays are rapid, automated, economical, and highly sensitive, consuming small
quantities of samples and reagents.[4] The concept and methodology of protein
microarrays was first introduced and illustrated in antibody microarrays (also referred
to as antibody matrix) in 1983 in a scientific publication [5] and a series of patents.
[6]

The high-throughput technology behind the protein microarray was relatively easy

to develop since it is based on the technology developed for DNA microarrays,

[7]

which have become the most widely used microarrays.

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Chapter 2
PROTEIN MICROARRAY TECHNOLOGY
Microarray technology allows the simultaneous analysis of thousands
ofparameters within a single experiment. Micro spots of capture molecules are
immobilized in rows and columns onto a solid support and exposed to samples
containing the corresponding binding molecules. Readout systems based on
fluorescence, chemiluminescence, mass spectrometry, radioactivity or electrochemistry
can be used to detect complex formation within each micro spot. Such miniaturized
and parallelized binding assays can be highly sensitive, and the extraordinary power
of the method is exemplified by array-based gene expression analysis. In these
systems, arrays containing immobilized DNA probes are exposed to complementary
targets and the degree of hybridization is measured. Recent developments in the field
of protein microarrays show applications for enzymesubstrate, DNAprotein and
different types of proteinprotein interactions.

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Chapter 3
MOTIVATION FOR DEVELOPMENT
Protein microarrays were developed due to the limitations of using DNA
microarrays for determining gene expression levels in proteomics. The quantity
of mRNA in the cell often doesnt reflect the expression levels of the proteins they
correspond to. Since it is usually the protein, rather than the mRNA, that has the
functional role in cell response, a novel approach was needed. Additionally posttranslational modifications, which are often critical for determining protein function,
are not visible on DNA microarrays.[8] Protein microarrays replace traditional
proteomics techniques such as 2D gel electrophoresis or chromatography, which were
time consuming, labor-intensive and ill-suited for the analysis of low abundant
proteins.

3.1 Making the Array

The proteins are arrayed onto a solid surface such as microscope slides,
membranes, beads or microtitre plates. The function of this surface is to provide a
support onto which proteins can be immobilized. It should demonstrate maximal
binding properties, whilst maintaining the protein in its native conformation so that its
binding ability is retained. Microscope slides made of glass or silicon are a popular
choice since they are compatible with the easily obtained robotic arrayers and laser
scanners that have been developed for DNA microarray technology.

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Fig 3.1. Micro Titre Plate

The chosen solid surface is then covered with a coating that must serve the
simultaneous functions of immobilising 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. In
addition, it also needs to display minimal non-specific binding in order to minimize
background noise in the detection systems. Furthermore, it needs to be compatible
with different detection systems. Immobilising agents include layers of aluminium or
gold,

hydrophilic

polymers,

with amines, aldehyde or epoxy.

and polyacrylamide
Thin-film

technologies

gels,

or

like physical

treatment
vapour

deposition (PVD) and chemical vapour deposition (CVD) are employed to apply the
coating to the support surface.
An aqueous environment is essential at all stages of array manufacture and
operation to prevent protein denaturation. Therefore sample buffers contain a high
percent of glycerol(to lower the freezing point), and the humidity of the manufacturing
environment is carefully regulated. Microwells have the dual advantage of providing
an aqueous environment while preventing cross-contamination between samples.
In the most common type of protein array, robots place large numbers of
proteins or their ligands onto a coated solid support in a pre-defined pattern. This is
known as robotic contact printing or robotic spotting.

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Fig 3.2.

Another fabrication method is ink-jetting, a drop-on-demand, non-contact

method of dispersing the protein polymers onto the solid surface in the desired pattern.
[9]

Piezoelectric spotting is a similar method to ink-jet printing. The printhead moves

across the array, and at each spot uses electric stimulation to deliver the protein
molecules onto the surface via tiny jets. This is also a non-contact process.
[10]

Photolithography is a fourth method of arraying the proteins onto the surface. Light

is used in association with photomasks, opaque plates with holes or transparencies that
allow light to shine through in a defined pattern. A series of chemical treatments then
enables deposition of the protein in the desired pattern upon the material underneath
the photomask.

The

capture

molecules

arrayed

on

the

solid

surface

may.be antibodies, antigens, aptamers (nucleic acid-based ligands),affibodies (small

molecules engineered to mimic monoclonal antibodies), or full length proteins.
Sources of such proteins include cell-based expression systems for recombinant
proteins, purification from natural sources, production in vitro bycell-free translation
systems, and synthetic methods for peptides. Many of these methods can be automated
for high throughput production but care must be taken to avoid conditions of synthesis

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or extraction that result in a denatured protein which, since it no longer recognizes its
binding partner, renders the array useless.

Proteins are highly sensitive to changes in their microenvironment. This

presents a challenge in maintaining protein arrays in a stable condition over extended
periods of time. In situ methods involve on-chip synthesis of proteins as and when
required, directly from the DNA using cell-free protein expression systems. Since
DNA is a highly stable molecule it does not deteriorate over time and is therefore
suited to long-term storage. This approach is also advantageous in that it circumvents
the laborious and often costly processes of separate protein purification and DNA
cloning, since proteins are made and immobilised simultaneously in a single step on
the chip surface. Examples of In situ techniques are PISA (protein in situ array),
NAPPA (nucleic acid programmable protein array) and DAPA (DNA array to protein
array).

Fig 3.3

Anumberofdifferentslidesurfacescanbeusedforproteinchips.Inchoosing
aslidesurface,thegoalsshouldbeimmobilizingtheproteinonthechip,maintaining
theconformationandthefunctionalityoftheprotein,andachievingmaximumbinding
capacity.Itisalsoimportanttoconsiderwhetherarandomorauniformorientationof
proteinsontheslidesurfaceisdesired(Figure2).Forrandomattachmentofproteins

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throughamines,aldehydeandepoxyderivatizedglasssurfacescanbeused.Coating
theglasssurfacewithnitrocellulose,gelpads,orpolyLlysinealsoachievesarandom
orientationoftheproteinsastheproteinsarepassivelyadsorbedontothesurface.
.

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Seminar Report 2015

Chapter 4
TYPES OF ARRAYS

There are three types of protein microarrays that are currently used to study the
biochemical activities of proteins.

4.1 Analytical microarrays

Analytical microarrays are also known as capture arrays. In this technique, a
library of antibodies, aptamers or affibodies is 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. Analysis of the
resulting binding reactions using various detection systems can provide information
about expression levels of particular proteins in the sample as well as measurements of
binding affinities and specificities. 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.

4.2 Functional protein microarrays

Functional protein microarrays (also known as target protein arrays) are
constructed by immobilising large numbers of purified proteins and are used to
identify protein-protein, protein-DNA, protein-RNA, protein-phospholipid, and
protein-small molecule interactions, to assay enzymatic activity and to detect

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antibodies and demonstrate their specificity. They differ from analytical arrays in that
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 the entire
proteome in a single experiment.
It is possible that the position of the affinity tags used for purification may
interfere with the functionality of the proteins. The use of two yeast proteome
collections, one with C-terminal tags, and one with a different N-terminal tag is
expected to complement each other version of the tagged protein exhibits a loss of
functionality due to the location of the tag, then the other tagged version of the protein
can be used on the chip.

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\
Recently a method has been described for the direct production of proteins on
chips. DNA is spotted on a microscope slide and subjected to an in vitro transcription
and translation system. The proteins are produced as GST fusions and adhere to

glutathione on the surface of the slide. Using this system LaBaer and coworkers have
produced a number of human proteins involved in DNA metabolism and demonstrated
protein-protein interactions (Ramachandran et al., 2004). This method is advantageous
because it produces protein directly on the slide without requiring purification and that
proteins do not need to be stored.

4.3 Reverse phase protein microarray (RPPA)

Reverse phase protein microarray (RPPA) involve complex samples, such as
tissue lysates. Cells are isolated from various tissues of interest and are lysed. The
lysate is arrayed onto the microarray and probed with antibodies against the target
protein of interest. These antibodies are typically detected with chemiluminescent,
fluorescent or colorimetricassays. Reference peptides are printed on the slides to allow
for protein quantification of the sample lysates. RPAs allow for the determination of
the presence of altered proteins or other agents that may be the result of disease.
Specifically, post-translational modifications, which are typically altered as a result of
disease can be detected using RPAs.

4.4 ProtoArray Human Protein Microarray

The ProtoArray Human Protein Microarray is an advanced, high-content,
functional protein microarray that enables rapid profiling of thousands of biochemical
interactions in as little as one day. Researchers can use the ProtoArray microarray to
perform a variety of assays including biomarker identification, drug target discovery,
enzyme substrate identification, antibody specificity profiling, and protein-protein
interaction studies. Results can be readily measured with most commercial scanners,

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and the data can be analyzed using our free ProtoArray Prospector software (Figure
1).

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Seminar Report 2015

Chapter 5.
DETECTION
Protein array detection methods must give a high signal and a low background.
The most common and widely used method for detection is fluorescence labeling
which is highly sensitive, safe and compatible with readily available microarray laser
scanners. Other labels can be used, such as affinity, photochemical or radioisotope
tags. These labels are attached to the probe itself and can interfere with the probetarget protein reaction. Therefore a number of label free detection methods are
available, such as surface plasmon resonance (SPR), carbon nanotubes, carbon
nanowire sensors (where detection occurs via changes in conductance) and
microelectromechanical system (MEMS) cantilevers. All these label free detection
methods are relatively new and are not yet suitable for high-throughput protein
interaction detection; however, they do offer much promise for the future.

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Chapter 6
APPLICATIONS
1. Diagnostics involves the detection of antigens and antibodies in blood samples;
the profiling of sera to discover new disease biomarkers; the monitoring of
disease states and responses to therapy in personalized medicine; the
monitoring of environment and food.
2. Proteomics pertains to protein expression profiling i.e. which proteins are
expressed in the lysate of a particular cell.
3. Protein functional analysis is the identification of protein-protein interactions
(e.g. identification of members of a protein complex), protein-phospholipid
interactions, small molecule targets, enzymatic substrates (particularly the
substrates ofkinases) and receptor ligands.
4. Antibody characterization is characterizing cross-reactivity, specificity and
mapping epitopes.Treatment development involves the development of
antigen-specific therapies for autoimmunity, cancer and allergies; the
identification of small molecule targets that could potentially be used as new
drugs.
5. The biochemistries of thousands of proteins can be characterized and
quantified in a parallel format through the use of protein microarrays. Not only
have protein chips been used to characterize the functions of previously
uncharacterized proteins, they have also been used to discover new
functionalities for previously characterized proteins.
6. Proteome chips have been used to study protein-protein interactions, proteinDNA interactions, protein-lipid interactions, protein-drug interactions, proteinreceptor interactions, and antigen-antibody interactions. In addition, proteome
chips have been used to study kinase activities and have been used for serum
profiling.

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Chapter 7.
PROTEIN CHIP PRECURSORS TO MODERN DAY

The equipment and reagents used in an Enzyme-linked Immunosorbent Assay

(ELISA), a precursor of protein chips.
Immunoassays, the precursor to protein chips available since the 1980s, exploit
the interactions between antibodies and antigens in order to detect their concentrations
in biological samples. Their creation, however, is tedious and expensive. As a response
to this, researchers at Harvard University combined the technologies of immunoassays
and DNA microarrays to develop the protein chip. [4] In their landmark paper, published
in 2000, "Printing Proteins as Microarrays for High-Throughput Function
Determination," Gavin MacBeath and Stuart Schreiber described how to create protein
chips and demonstrated three types of applications that would benefit from this new
technology. The strengths of their approach were the use of readily available materials
(i.e. glass slides, polyacrylamide gel), the relative ease of implementation (robotic
microarray printers), and compatibility with standard instrumentation.
Within the past five years, many companies, including Biacore, Invitrogen,
and Sigma-Aldrich, have begun production of industrial level protein array systems
that can be used for drug discovery and basic biological research. Commercial entities
have made protein chip research a streamlined and standardized process on the same
level as DNA microarrays compared to its inception in 2000.

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Chapter 8.
CHALLENGES
Despite the considerable investments made by several companies, proteins
chips have yet to flood the market. Manufacturers have found that proteins are actually
quite difficult to handle. A protein chip requires a lot more steps in its creation than
does a DNA chip.

1. Finding a surface and a method of attachment that allows the proteins to

maintain their secondary or tertiary structure and thus their biological activity
and their interactions with other molecules.
2. Producing an array with a long shelf life so that the proteins on the chip do not
denature over a short time.
3. Identifying and isolating antibodies or other capture molecules against every
protein in the human genome
4. quantifying the levels of bound protein while assuring sensitivity and avoiding
background noise.
5. Extracting the detected protein from the chip in order to further analyze it.
6. Reducing non-specific binding by the capture agents.
7. The capacity of the chip must be sufficient to allow as complete a
representation of the proteome to be visualized as possible; the detection of less
abundant proteins such as signaling molecules and receptors, which are
generally of more therapeutic interest.

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Seminar Report 2015

Chapter 9
ADVANTAGES

1.
2.
3.
4.
5.
6.

Can screen many proteins simultaneously

Small amounts of proteins and reagents
High throughput
Diverse applications-biochemical assays, posttranslational modifications, small
molecule screening
In Vitro Assay

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Seminar Report 2015

Chapter 10.
CONCLUSION
In the past, studies of protein activities have focused on studying a single
protein at a time, which is often time-consuming and expensive. Recently, with the
sequencing of entire genomes, large-scale proteome analysis has begun. Arrays of
proteins have been used for the determination of subcellular localization, analysis of
proteinprotein interactions and biochemical analysis of protein function. New
protein-microarray technologies have been introduced that enable the high-throughput
analysis of protein activities. These have the potential to revolutionize the analysis of
entire proteomes.

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Seminar Report 2015

REFERENCES
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microarrays:

comparison

of

current

microarray

support

materials. Anal

Biochem. 2002;309:25360. [PubMed]

2. Bertone P, Snyder M. Advances in functional protein microarray technology. Febs
J. 2005;272:540011. [PubMed]
3. Brasch MA, Hartley JL, Vidal M. ORFeome cloning and systems biology:
standardized

mass

production

of

the

parts

from

the

parts-list. Genome

Res. 2004;14:20019. [PubMed]

4. Brizuela L, Braun P, LaBaer J. FLEXGene repository: from sequenced genomes to
gene

repositories

for

high-throughput

functional

biology

and

proteomics. MolBiochemParasitol. 2001;118:15565.[PubMed]

5. Charles PT, Goldman ER, Rangasammy JG, Schauer CL, Chen MS, Taitt CR.
Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity
and

antibody

functionality

for

biosensor

applications. BiosensBioelectron. 2004;20:75364. [PubMed]

6. Colca JR, Harrigan GG. Photo-affinity labeling strategies in identifying the protein
ligands of bioactive small molecules: examples of targeted synthesis of drug analog
photoprobes. Comb Chem High Throughput Screen. 2004;7:699704. [PubMed]