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Protein Arrays Resource Page
review | literature | meetings | companies

Introduction  
Array Basics Defining characteristics  
  Types of protein arrays  
Areas of application  
Protein sources  
Formats and surfaces  
Prtotein immobilisation considerations  
Fabrication  
Detection  
Capture arrays Binding molecules  
  Detection systems  
  Specificity and cross-reactivity  
Related formats  
  Applications of capture arrays Diagnostics
    Expression profiling
Protein arrays Protein in-situ arrays PISA
NAPPA
DAPA
Protein functions and interactions  
Library screening  
Challenges/Bottlenecks Protein production and function  
  Capture reagents and cross-reactivity  
Array technology  
Literature Guide Original papers  
Reviews  
Meetings  
Companies  

Introduction

Protein arrays are rapidly becoming established as a powerful means to detect proteins, monitor their expression levels, and investigate protein interactions and functions. They are seeing an explosive progress and interest at the moment and have become one of the most active areas emerging in biotechnology today. The objective behind protein array development is to achieve efficient and sensitive high throughput protein analysis, carrying out large numbers of determinations in parallel by automated means. While they were conceived originally as miniaturised immunoassays, further development is being driven by genome projects on the one hand and improved expertise in expression of recombinant proteins on the other. Protein arrays make possible the parallel multiplex screening of thousands of interactions, encompassing protein-antibody, protein-protein, protein-ligand or protein-drug, enzyme-substrate screening and multianalyte diagnostic assays. In the microarray or chip format, such determinations can be carried out with minimum use of materials while generating large amounts of data. Moreover, since most proteins are made by recombinant methods, there is direct connectivity between results on protein arrays and DNA sequence information.

At the present time, protein arrays are poised to become a central proteomics technology, important both in basic research and commercially for biotechnology enterprises. It is well recognised that the complexity of the human proteome far exceeds that of the genome. When variables such as alternative gene splicing events, post-translational modifications and individual coding variants are taken into account, the number of different molecular protein species in man is likely to be at least 1-2 orders of magnitude greater than the number of genes (about 21,000), i.e. possibly as many as 1,000,000 proteins. Proteomics investigations are at the leading edge of functional genomics today and the development of protein arrays reflects the realisation that functional genomics discoveries will depend heavily on progress in defining the expression of, and interactions among, proteins. Conventional proteome analysis by 2D gel electrophoresis and mass spectrometry, while highly effective, has limitations and in particular may miss many proteins of interest when expressed at low abundance and is unsuited to diagnostic applications . Since the low abundance proteins are often those of the greatest diagnostic interest (e.g. cytokines and biomarkers in plasma), there is therefore an acknowledged need for other highly sensitive, specific and accessible high throughput technologies for protein detection, quantitation and differential expression analysis in health and disease. For this reason, protein arrays are generating enormous interest at the research and biotechnology levels.

Microarray ELISA-style assays will accelerate immunodiagnostics significantly. This aspect of the technology was discussed in the 1980s by Ekins, who introduced the concept of the ambient analyte assay and demonstrated that microspot immunoassays could be perfomed with high sensitivity and selectivity. In addition to diagnostics applications, protein array technology promises to accelerate basic research on protein-protein interactions and will allow protein expression profiling, ranging from limited numbers of proteins up to global proteomic analysis, while in the pharmaceutical industry protein arrays can be integrated into target identification and validation processes. However, as with other high throughput functional genomics technologies, there are major technical demands which will need to be solved in order to achieve maximum capability. This page will attempt to keep abreast of developments in protein array technologies and to provide an up to date guide to the relevant literature, meetings and companies in the field.

(Note, in the text below, mention is made of specific companies developing protein arrays; links to their websites can be found on the companies page. While specific literature references are not annotated in the text, a comprehensive literature list is provided.)


Array basics

Defining characteristics of protein arrays
Protein arrays are solid-phase ligand binding assay systems using immobilised proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The assays are highly parallel (multiplexed) and often miniaturised (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. Fortunately some of the software can be adapted from that used for DNA arrays, as can much of the hardware and detection systems.

 

 

 

 

 

 

 

 

 

Types of protein arrays
There are three general types of protein array:
(a) large-scale functional chips ( target protein arrays ) constructed by immobilising large numbers of purified proteins and used to assay a wide range of biochemical functions, such as protein-protein, protein-DNA, protein-small molecule interactions and enzyme activity, and to detect antibodies and demonstrate their specificity.
(b) the analytical capture arrays carry affinity reagents, primarily antibodies, but may also be alternative protein scaffolds, peptides or nucleic acid aptamers, and are used to detect and quantitate analytes in complex mixtures such as plasma/serum or tissue extracts;
(c) lysate (reverse protein) arrays in which the complex samples – such as tissue lysates - are printed on the surface and targets then detected with antibodies overlaid on them.

 

 

 

 

 

 

 

 

 

 

 

Areas of application
All three array types have uses in diagnostics (biomarkers or antibody detection) and discovery research. T he capture arrays, are used to detect target molecules in mixtures such as plasma or tissue extracts. Typically a complex mixture would be applied to arrays of possibly thousands of specific binders, and the individual bound analytes detected in parallel by appropriate labelling and scanning. In diagnostics, capture arrays can be used to carry out multiple immunoassays in parallel, both testing for several analytes in individual sera for example and (by segmenting the array) testing many serum samples simultaneously. In proteomics, capture arrays are used to quantitate and compare the levels of proteins in different samples in health and disease, i.e. protein expression profiling.
Target protein arrays are used in for in vitro functional interaction screens and particularly to detect antibodies in individual patient or animal sera, during disease or to monitor immune responses. The capture reagents themselves will need to be selected and screened for cross reactivity against many proteins, which can also be done in a multiplex array format against multiple protein targets.
Lysate arrays allow thousands of samples to be analysed simultaneously on the same platform, greatly increasing throughput and simplifying quantitative analysis between samples. Furthermore, an exceedingly small amount of sample is required for printing the arrays, thus permitting the analysis of rare and valuable patient samples.

Thus, broadly speaking, there are at least four major areas where protein arrays are being applied, each of which requires appropriate formats and readout methods:
1. Diagnostics: detection of antigens and antibodies in blood samples; profiling of sera to discover new disease markers; environment and food monitoring. Applications in autoimmunity, allergy and cancer are listed in the tables below.
2. Proteomics: Protein expression profiling
3. Protein functional analysis: protein-protein interactions; ligand-binding properties of receptors; enzyme activities;
4. Antibody characterisation: cross reactivity and specificity, epitope mapping.














 

 

 

 

 

 


Protein sources

For construction of arrays, sources of 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. For capture arrays and protein function analysis, it is clearly important that proteins should be correctly folded and functional; this is not always the case, e.g. where recombinant proteins are extracted from bacteria under denaturing conditions or where the surface encourages unfolding . On the other hand, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, identifying autoantibodies and selecting ligand binding proteins , where linear epitopes are recognised .

Formats and surfaces
Protein arrays have been designed as a miniaturisation of familiar immunoassay methods such as ELISA and dot blotting, often utilising fluorescent readout, and facilitated by robotics and high throughput detection systems to enable multiple assays to be carried out in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While microdrops of protein delivered onto planar surfaces are the most familiar format, there are a number of more advanced architectures incorporating developments in microfluidics and nanotechnology. Particles in suspension can also be used as the basis of arrays, providing they are coded for identification [e.g. Luminex, Bio-Rad systems].

Protein immobilisation considerations
Variables in immobilisation of proteins include both the coupling reagent and the nature of the surface being coupled to. The properties of a good protein array support surface are that it should be chemically stable before and after the coupling procedures; allow good spot morphology; display minimal nonspecific binding; not contribute a background in detection systems; and be compatible with different detection systems. The immobilisation method used should be reproducible; applicable to proteins of different properties (size, hydrophilic, hydrophobic); amenable to high throughput and automation; and compatible with retention of fully functional protein activity. Orientation of the surface-bound protein is recognised as a possible factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labelling of the protein.

Both covalent and noncovalent methods of protein immobilisation are used and have various pros and cons. Diffusion into porous surfaces is a successful method, allowing noncovalent binding of unmodified protein within hydrogel structures, based on a 3-dimensional polyacrylamide gel; these substrates are reported to give a particularly low background on glass microarrays, with a high capacity and retention of protein function. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientational control; it may alter the functional properties of the protein through unfolding, and reproducibility and efficiency are variable. Covalent coupling methods provide a stable linkage, can be applied to a range of proteins and have good reproducibility; however, orientation may be variable, chemical derivatisation may alter the function of the protein and requires a stable interactive surface. Biological capture methods utilising a tag (such as hexahistidine/Ni-NTA or biotin/avidin) on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the partner reagent must first be immobilised adequately on the surface.

 

 

 

 

 

 

 

 

 

Fabrication
Array fabrication methods include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography. A number of commercial arrayers (spotters) are available as well as manual equipment. The novel methods for production of protein arrays in situ (see section below) avoid the need to express and purify the proteins, spotting instead the DNA templates from which proteins are expressed on the array itself from cell free transcription/translation systems.

 

 

 

 

 

 

 

 

 

 

At the limit of spot size and density are nanoarrays, with spots on the nanometer spatial scale, enabling thousands of reactions to be performed on a single chip less than 1mm square.

Detection
Fluorescence labelling and detection methods are widely used and are highly sensitive. The same instrumentation as used for scanning DNA microarrays is applicable to protein arrays. For differential display, capture (e.g. antibody) arrays can be probed with fluorescently labelled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the colour acts as a readout for changes in target abundance. Use of 2-colour assays with direct labelling allows control for spot variability as well as comparison between two different samples. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA). Planar waveguide technology enables ultrasensitive fluorescence detection, with the additional advantage of no intervening washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label or the properties of semiconductor nanocrystals (Quantum dots). A number of novel alternative readouts have been developed, including adaptations of surface plasmon resonance (for real time kinetic measurements), rolling circle DNA amplification (for sensitivity down to near the single molecule level), mass spectrometry (for definitive protein identification), resonance light scattering and atomic force microscopy (for nanoarrays).

 

 

 

 

 

 

 

 

 

Capture arrays

These form the basis of diagnostic chips for detection of clinically important ligands, such as biomarkers, and arrays for expression profiling. They employ high affinity capture reagents, including conventional and recombinant (single chain) antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput, parallel manner.

Binding molecules
Antibody arrays have the required properties of specificity and acceptable background, and a number are available commercially for different sets of antigens, such as cell signalling pathways. Antibodies for capture arrays are made either by conventional immunisation (polyclonal sera and hybridomas), or as recombinant single chain fragments (scFv), usually expressed in E. coli, after selection from phage or ribosome display libraries. In addition, single V-domains from camelids or engineered human equivalents may also be useful in arrays.

The term 'scaffold' refers to protein domains which are engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity. The variants can be produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display. Such binding scaffolds or frameworks include Designed Ankyrin Repeat proteins (DARPins), Affibody molecules based on Staph. aureus protein A, Trinectins based on fibronectins and Anticalins based on the lipocalin structure. These can be used on capture arrays in a similar fashion to antibodies and often have advantages of robustness and ease of production.

Nonprotein capture molecules, notably the single-stranded nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays. Aptamers are selected from libraries of oligonucleotides by the Selex™ procedure and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand reduces the crossreactivity of aptamers due to the specific steric requirements. More recently, SomaLogic introduced ‘Slow’ Aptamers or ‘SLaptamers’, selected for a slow rate of dissociation (t1/2 >30mins), of which several hundred specificities hacve now been selected. Aptamers in general have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA.

Detection systems for capture arrays

 

 

 

 

 

 

 

 

What is required from any method is optimal sensitivity and specificity, with low background to give high signal to noise. Since analyte concentrations cover a wide range, sensitivity has to be tailored appropriately; serial dilution of the sample or use of antibodies of different affinities are solutions to this problem. Sensitivity is particularly important where the proteins of interest are in very low concentration in body fluids and extracts, requiring detection in the pg range or lower, such as cytokines or the low expression products in cells.
Protein analytes binding to antibody arrays may be detected directly or via a secondary antibody in a sandwich assay. Direct labelling is used for comparison of different samples with different fluorophores. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label-free detection methods, including MS and SPR, avoid alteration of ligand.

Specificity and cross-reactivity on capture arrays
The question of cross-reactivity is an important one which applies to all ligand binders and particularly to antibodies, being the most popular reagents. While antibodies are thought of as being highly specific, monoclonals can show unpredictable cross-reactions which will be revealed by thorough screening. The ultimate usefulness of individual reagents then depends on the relative level of cross-reaction and specific reaction. An important general principle is that, for optimal specificity where assays are highly multiplexed, it is essential to provide dual level target recognition, i.e. two levels of specificity for each locus in the array. Sandwich assays achieve this with two antibodies, photocrosslinking reduces the cross-reactivity of aptamers and MS provides definitive label-free protein identification.

The use of sandwich assays (above), in which antibody pairs are used to bind and detect ligand, go a long way towards eliminating the problem, since it is unlikely that both members of the sandwich will exhibit the same cross-reactivity. Nevertheless, the multiplexing of sandwich assays is limited to around 40 reactions, again due to cross-reactivity of the reagents. A related technology in which pairs of binders recognise the same target protein is the proximity ligation method which, combined with rolling circle amplification, can be adapted for highly sensitive and specific protein assay in the array format.

Related formats
Another methodology which can be used diagnostically and in expression profiling is the ProteinChip® array [Ciphergen], in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumour extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins. The ProteinChip® is credited with the ability to identify novel disease markers. However, this technology differs from the protein arrays under discussion here since, in general, it does not involve immobilisation of individual proteins for detection of specific ligand interactions.

Applications of capture arrays

1. Diagnostics
As diagnostic devices, microarrays exploit the power of multiplexing simultaneous analyses of different samples and repeated analyses of the same samples. Diagnostics formats include arrays of antibodies, as in detection of cytokines [e.g. Zyomyx, Zeptosens, Molecular Staging, Luminex], and antigens to detect serum antibodies in screens for infections, autoimmune diseases and allergies. Highly parallel analysis on arrays will allow determination of tumour markers in extracts with only a minimum of biopsy material, creating new possibilities for monitoring cancer treatment and therapy. Discovery of new autoantibody specificities is possible by screening patient sera against arrays of human proteins [Protagen].
While diagnostic arrays have tended to be of relatively low density and designed for specific assay purposes, they have high throughput potential through automated image analysis and microfluidics.

2. Proteomics: Protein expression profiling
The quantitative detection of proteins in cells and tissues and comparison in different conditions (health, disease, differentiation, drug treatment, etc) is a central aim of proteomics. The array format is well established for the rapid, global analysis of nucleic acids, as in the use of oligonucleotide and cDNA arrays for gene expression (transcription) profiling. However, mRNA expression data has acknowledged shortcomings as an indicator of actual protein abundance or dynamics, and moreover reveals nothing about post-translational modifications or protein-protein interactions. Two-dimensional gel electrophoresis technology, on which most proteome profiling is based currently, is also limited in various ways, particularly in the difficulty of finding and quantitatively estimating low abundance proteins. For information about the expression of the proteome, protein and peptide arrays are becoming major tools and the information that will be obtained from them in the future will complement transcriptional data. Capture arrays sensitively and accurately detect low levels of proteins with minimal technical know-how on the part of the user and we can expect them to be used widely to measure differential protein expression. They will provide a powerful and reliable platform for extending molecular analysis beyond the limitations of DNA chips. This assumes that the necessary numbers of antibodies or other capture reagents of required specificity and affinity can be obtained against the proteins of interest - at the limit, against the entire proteome. There are ambitious binder project projects in the planning stage to fill this need (e.g. ProteomeBinders).

 

 

 

 

 

 

 

 

 

 

 

A format for differential protein expression profiling using antibody arrays is shown in the figure above. A mixture (e.g. of two tissue extracts) is applied to the array and the analytes of interest are captured by the specific ligand binders, followed by detection of binding. Similar to comparison of samples from normal and diseased tissues on DNA arrays or on 2D gels, reference and test samples can be labelled with Cy3 and Cy5 fluors, mixed, gel filtered to remove unbound dyes and then incubated on a chip of arrayed antibodies. Increased or decreased protein expression is assessed using a scanner and up- or down-regulated proteins can be identified from the ratios of the two dyes in the familiar 'traffic light' (red, yellow, green) system. Here, directly labelled (covalently derivatised) samples are used, but there are a number of alternative detection strategies.

Protein arrays

Functional chips have been constructed by immobilising large numbers of purified proteins and used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrates, etc. Generally they require an expression library, cloned into E. coli, yeast or similar from which the expressed proteins are then purified, e.g. via a His tag, and immobilised. Cell free protein transcription/translation is a valuable alternative for synthesis of proteins which do not express well in bacterial or other in vivo systems.

In situ protein arrays

Due to the sensitivity and hetergeneity of proteins, it is difficult to store large- scale protein arrays in a functional state for long periods of time. In contrast, DNA is a highly stable molecule capable of long-term storage. Therefore, an interesting concept is to make protein arrays directly from DNA, either co-distributed or pre-arrayed, using cell free protein experssion systems, to create the proteins on the arrays on demand as and when required. Moreover, since the proteins are made and immobilised simultaneously in a single step on the chip surface, the laborious and often costly processes of separate protein purification and DNA cloning are avoided.

Thus, in situ (‘on-chip’) methods address three issues in protein array technology: (i) efficient global protein expression and availability; (ii) functional protein immobilisation and purification; and (iii) on-chip stability over time. Three recently described methods are termed PISA (protein in situ array), NAPPA (nucleic acid programmable protein array) and DAPA (DNA array to protein array).

PISA method
In PISA (He, M., Taussig M.J. Nucleic Acids Res 29, e73, 2001), proteins are made directly from DNA, either in solution or immobilised, and become attached to the array surface as they are made, through recognition of a tag sequence. The proteins are expressed in parallel in vitro utilising a cell free system, commonly rabbit reticulocyte or E. coli S30, to perform coupled transcription and translation. The key feature of the method is that protein expression is performed on a surface which is precoated with an immobilising agent capable of binding the tag. Thus after each protein is translated, it becomes fixed simultaneously and specifically to the surface and the other material can be washed away. Starting from PCR DNA, the PISA procedure takes about 3-4 hours to create the protein array. Microarrays are produced directly onto glass slides, either by mixing the DNA with the cell free lysate system before spotting or by a multiple spotting technique (MIST) in which DNA is spotted first followed by the expression system (Angenendt P. et al, 2006).

 

 

 

 

 

 

 

 

 

NAPPA method
Transcription and translation from an immobilised (as opposed to a solution) DNA template is a further desirable development of on-chip technologies which would allow conversion of DNA arrays to protein arrays. This has been initiated with the system called Nucleic Acid Programmable Protein Array or NAPPA (Ramachandran N. et al. Science 305, 86-90, 2004). Biotinylated cDNA plasmids encoding the proteins as GST fusions are printed onto an avidin coated slide, together with an anti-GST antibody acting as the capture entity. The cDNA array is then covered with rabbit reticulocyte lysate to express the proteins, which become trapped by the antibody adjacent to each DNA spot, the proteins thereby becoming immobilised with the same layout as the cDNA. This procedure was shown to work quite precisely, with discrete protein spots and minimal diffusion or cross-talk, and used for functional studies of interactions between cell cycle proteins. Recently it was expanded to high density arrays of 1000 different proteins (Ramachandran et al. Nature Methods 5:535-538, 2008). Note that this technology generates a protein array in which the immobilised proteins are present together with DNA and capture agent, and furthermore that the DNA array can only be used once.

 

 

 

 

 

 

DAPA method
This method for in situ protein arraying represents a further advance in that it uses an immobilised DNA array as the template to generate ‘pure’ protein arrays on a separate surface from the DNA, and also is able to produce multiple copies of a protein array from the same DNA template (He M, et al. Nature Methods, 5, 175-7, 2008). Cell-free protein synthesis is performed in a membrane held between two surfaces (glass slides), one of which is arrayed with DNA molecules while the other surface carries a specific reagent to capture the translated proteins. Individual, tagged proteins are synthesised in parallel from the arrayed DNA, diffuse across the gap and subsequently immobilised through interaction with the tag-capturing reagent on the opposite surface to form a protein array. Discrete spots which accurately reflect the DNA in position and quantity are produced. Moreover replicate copies of the protein array can be obtained by reuse of the DNA, and at least 20 repeats have been demonstrated.

 

 

 

 

 

 

 

 

 

 

 

 

 

Screening for protein functions and interactions
For detecting protein-protein interactions, protein arrays can be in vitro alternatives to the cell-based yeast two-hybrid system and may be useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulphide bridges. High-throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of the yeast proteome, where a large proportion of all yeast open-reading frames was expressed and immobilised on a microarray. Large-scale 'proteome chips' promise to be very useful in identification of functional interactions, drug screening, etc. Another possible screen will be for the effect of polymorphisms arising from disease-related coding SNPs (SAPs, single amino acid polymorphisms); such information may be valuable in ascertaining the effects of SNPs on drug responses and side effects in patients (pharmacogenomics). One restriction is that proteins which are only functional as multicomponent complexes will be more difficult to analyse on protein arrays.

Library screening
As a two-dimensional display of individual elements, a protein array can be used to screen phage or ribosome display libraries, in order to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, 'library against library' screening can be carried out. Screening of drug candidates in combinatorial chemical libraries against an array of protein targets identified from genome projects is another application of the approach.


Remaining Challenges and bottlenecks

Despite the recent progress, there are a number of important technical challenges and bottlenecks in protein array technologies, some of which are unique to proteins while others are common to high throughput methods in general, which will need to be solved in order to achieve the maximum capability. They include the problems of obtaining global, functional protein expression for array construction and selection of protein binders, aspects of protein coupling to surfaces, the sensitivity and dynamic range of detection systems, and standardisation and data storage. At each bottleneck there are choices of alternative methods. Issues which need to be addressed include the following.

Protein production and function
A bottleneck in creating protein arrays, especially those which aim to be global, is the production (expression and purification) of the huge diversity of proteins which will form the array elements, including capture molecules. Collections of high-quality expression clones for different species are required and systems for the production of proteins in high throughput must be developed. The challenge will be to make expression methods sufficiently comprehensive such that potentially all proteins (including membrane proteins) become available; different systems (bacterial, yeast, baculovirus and cell-free) will be employed for different proteins. One aim will be to express many of the proteins for functional analysis, and another is to raise antibodies and other capture reagents against them for array production.

A further consideration for many purposes is that arrayed proteins should be correctly folded and functional. This will require extensive and almost individual validation, which with proteins of unknown function may be hard to achieve! Production and functional immobilisation of membrane proteins, which comprise a large proportion of the proteome, is a continuing difficulty; these may be best accessed in the new cell arrays. Proteome chemistry is also hugely complicated by the existence of frequent and varied post-translational modifications (PTMs). The problem will be how to incorporate PTMs, of which phosphorylation and glycosylation are just two of many, into protein arrays. As well as wanting to put modified proteins onto the chip surface, we would also like to know the PTMs of captured proteins, which can be determined through PTM-specific antibodies or mass spectrometry.

Capture reagents and cross-reactivity
A current limitation on capture arrays is the availability of the antibodies (or scaffold proteins, etc.), especially where pairs are required against each target for sandwich assays. Accessing very large numbers of affinity reagents is a major challenge. Automated screening of antibody or scaffold libraries against arrays of target proteins may be the most rapid way of developing the thousands of reagents required for proteome expression profiling. There is some discussion over whether polyclonal antisera, hybridomas or selection from recombinant library systems is the best way forward, but in practice, providing they are screened thoroughly for cross-reactivity (below), products of all three are used successfully in the array format.

The design of capture arrays, particularly when exposed to heterologous mixtures such as plasma and tissue extracts, needs to take into consideration the problems of cross-reactivity which will occur particularly with highly multiplexed assays. Antibodies can be surprisingly cross-reactive, which in the high throughput microarray field can render results misleading or, at worst, useless. Successful multianalyte analysis will therefore require careful screening of each polyclonal antiserum, hybridoma or recombinant clone for cross-reactions against all antigens on the array. The use of combinations of antibodies against individual targets in sandwich assays should help to minimise cross-reactions. The importance of dual level target recognition, ensuring two levels of specificity for each element in the array, has been noted. This can be achieved through combinations of antibodies against individual targets in sandwich assays or proximity ligation, or through linkage to mass spectrometry to confirm the identity of bound ligands.

Array technology
What are the best coupling chemistries and supports? There are several options available in both categories (see Table above). Comparisons of different systems are gradually becoming available. The stability and lifetime of protein arrays in different formats needs to be considered; protein arrays are likely to be far less robust than DNA arrays.

Detection methods are another important consideration, with requirements of sensitivity, accuracy and quantitation over a wide range. The design of the array will be influenced by the readout system.

Finally, standardisation is an issue common to all high throughput technologies: the existence and development of many alternative formats and conditions inevitably leads to problems in comparison of results. Standards for protein arrays and a framework for their implementation will need to be established at an international level.

Comments on this webpage and suggestions for improvement are welcome. Please e-mail them to Mike Taussig.
Page last updated 20 May 2009.

© Mike Taussig & Oda Stoevesandt, 2009