Protein microarray

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.

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
doesn't 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 post-translational 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.

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. Nitrocellulose film
slides are broadly accepted as the highest protein binding substrate for protein microarray
applications.

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. 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, and polyacrylamide gels, or treatment with amines, aldehyde or
epoxy. Thin-film technologies like physical 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. 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.[11]

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 by cell-
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 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 —
invented and published by Mingyue He and Michael Taussig in 2001[12][13] — 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).

Types of arrays

Types of protein arrays

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

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.

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 colorimetric assays. 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.[14]

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 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.

The key element in any functional protein microarray-based assay is the arrayed proteins must
retain their native structure, such that meaningful functional interactions can take place on the
array surface. The advantages of controlling the precise mode of surface attachment through
use of an appropriate affinity tag are that the immobilised proteins will have a homogeneous
orientation resulting in a higher specific activity and higher signal-to-noise ratio in assays, with
less interference from non-specific interactions.[15][16]

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 probe-target 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.[17] 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. Immunoassays on thiol-ene "synthetic
paper" micropillar scaffolds have shown to generate a superior fluorescence signal.[18]

Protein quantitation on nitrocellulose coated glass slides can use near-IR fluorescent detection.
This limits interferences due to auto-fluorescence of the nitrocellulose at the UV wavelengths
used for standard fluorescent detection probes.[19]

Applications

There are five major areas where protein arrays are being applied: diagnostics, proteomics,
protein functional analysis, antibody characterization, and treatment development.

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. Digital bioassay is an
example of using protein microarray for diagnostic purposes. In this technology, an array of
microwells on a glass/polymer chip are seeded with magnetic beads (coated with fluorescent
tagged antibodies), subjected to targeted antigens and then characterised by a microscope
through counting fluorescing wells. A cost-effective fabrication platform (using OSTE polymers)
for such microwell arrays has been recently demonstrated and the bio-assay model system has
been successfully characterised.[20]
Proteomics pertains to protein expression profiling i.e. which proteins are expressed in the
lysate of a particular cell.

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 of kinases) and receptor ligands.

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.

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.
Production of reliable, consistent, high-throughput proteins that are correctly folded and
functional is fraught with difficulties as they often result in low-yield of proteins due to
decreased solubility and formation of inclusion bodies. A protein chip requires a lot more steps
in its creation than does a DNA chip.

There are a number of approaches to this problem which differ fundamentally according to
whether the proteins are immobilised through non-specific, poorly defined interactions, or
through a specific set of known interactions. The former approach is attractive in its simplicity
and is compatible with purified proteins derived from native or recombinant sources[21][22] but
suffers from a number of risks. Most notable amongst these relate to the uncontrolled nature of
the interactions between each protein and the surface; at best, this might give rise to a
heterogeneous population of proteins in which active sites are sometimes occluded by the
surface; at worst, it might destroy activity altogether due to partial or complete surface-mediated
unfolding of the immobilised protein.

Challenges include: 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; abundant proteins overwhelm the
detection of less abundant proteins such as signaling molecules and receptors, which are
generally of more therapeutic interest.[23]

See also

Microarray imprinting and surface energy patterning

Label-Free Detection techniques for protein microarrays (https://onlinelibrary.wiley.com/doi/fu

ll/10.1002/pmic.200900458)

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