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Microarray technology: an increasing variety of screening tools for proteomic research

Dieter Stoll, Jutta Bachmann, Markus F.Templin and Thomas O. Joos
Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment.Assay systems based on this technology are currently used for the identification, quantitation and functional analysis of proteins that are of interest for proteomic research in basic and applied biology and for diagnostic applications.Such novel assays are also of major interest for the pharmaceutical industry,focusing on the identification of biomarkers and the validation of potential target molecules.Sensitivity,reproducibility, robustness and automation have to be demonstrated before this technology will be suitable for high-throughput applications.
Dieter Stoll Markus F.Templin Thomas O. Joos* NMI Natural and Medical Sciences Institute at the University of Tuebingen Markwiesenstr. 55, 72770 Reutlingen, Germany Jutta Bachmann Hagelundveien 44 1450 Nesoddtangen Norway *e-mail: joos@nmi.de

w The rapid progress of whole genome sequencing in combination with well-established DNA microarrays and sophisticated bioinformatics platforms allows scientists to take a global view into biological systems (http:// www.genomenewsnetwork.org). In todays proteome era, it is time for protein microarrays to be used to screen entire genomes for proteins that interact with particular factors, catalyze particular reactions, or act as substrates for protein-modifying enzymes and/or as targets of autoimmune responses [1,2]. Antibody arrays and miniaturized multiplexed sandwich immunoassays enable scientists to measure protein concentrations quickly and simultaneously. As a result of miniaturization, microarrays can analyze many parameters in parallel and only require minimal amounts of reagents and sample. In this review we will summarize the current state of microarraybased approaches for proteome research, specifically focusing on applications in the drug discovery process.

Challenges for protein microarrays

In proteome research there is a huge demand for elucidating the function of proteins within

their cellular context. Protein functions are not only regulated by the presence or absence of distinct proteins but also by post-translational modifications and proteolytic protein processing resulting in changes in compartmentalization or in the interactions and formation of complexes of specific proteins. Microarray technology has a great potential to identify and quantify proteins and to study protein function from a global perspective [1,2]. Several different technologies are currently used to analyze the complex and highly dynamic protein content of biological systems. Unfortunately, none of them have the power to tackle the whole complexity and the dynamic range (estimated at 107) of expressed proteins in a cell, ranging from very low numbers of transcription factors up to millions of copies of certain types of cytoskeleton molecules [3]. 2D-PAGE is still the most widely used separation technology for the simultaneous examination of large numbers of proteins. However, 2D-PAGE suffers from low-sample throughput, small dynamic detection range and several unresolved problems in analyzing hydrophobic, small, and very basic or acidic proteins. Approaches involving multidimensional chromatography (MudPIT [4]) and different affinity capture chromatographic methods (e.g. ICAT [5]) were used as promising alternatives before mass spectrometry-based protein identification. But despite the very impressive developments of technologies used for proteome analysis within the last few years, the need for alternative technologies is still evident. The basic principles of miniaturized multiplexed ligand-binding assays were already described and discussed for proteins in the early 1980s [6]. Roger Ekins showed that small amounts of capture probes were mandatory

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for detecting correct analyte concentrations with the highest possible accuracy and sensitivity in analyte capture assays. Currently, microspot capture assays are on the way to becoming important tools in protein profiling. Compared to the well-established DNA microarray technology, however, the application of protein microarrays to the study of proteomes is still not very advanced. [2,7]. Hundreds of companies and technology providers are on the market offering products such as microarray surfaces (recently summarized in Zhu et al. [8]), arrayers and detection systems. However, the number of companies offering proteinmicroarray-related products that go beyond the basic technological aspects is still a mere handful (Table 1 and www.biochipnet.de).

In contrast to 2D-PAGE and mass-spectrometry-based proteome analysis methods, the design of specific capture arrays depends on the target proteins and the availability of highly specific capture agents against them. The limited availability of highly specific capture molecules and their unpredictable functionality are the main differences between DNA and protein microarray technologies. DNA is a rather uniform molecule and binds its complementary targets according to the well-defined base-pairing principle. It is very easy to predict highly selective and specific DNA capture sequences from the primary sequence of the target DNA. In addition, high-throughput oligonucleotide synthesis facilities or PCR-based approaches enable the fast and cost-effective generation of DNA capture agents.
a

Table 1. Examples of commercially available miniaturized and parallelized protein assays

Planar protein arrays Company Schleicher & Schuell Bioscience Zyomyx Inc. Pierce Biotechnology Inc. RayBiotech Inc. BD Biosciences SIGMA-ALDRICH Co. Protometrix, Inc. Molecular Staging, Inc. Zeptosens AG Ciphergen Biosystems Inc Bead-based systems Company/Technology Luminex Corporation

Product ProVision HCA Zyomyx Protein Profiling Biochip System SearchLight Arrays RayBio Cytokine Arrays, Custom Ab Arrays, BD Clontech Ab Microarray Panorama Ab Microarray Cell Signalling Kit The Yeast ProtoArray Rolling circle amplification technology (RCAT) ZeptoMAR CeLyA Cell Lysate Arrays SELDI Protein Chip technology

Applications/Kits Cytokine profiling Cytokine profiling Cytokine profiling Cytokine and protein profiling Comparative protein analysis Comparative protein analysis Services protein interaction studies Multiplexed protein profiling Reverse screening Reverse screening

Web link www.schleicher-schuell.de/ bioscience www.zyomyx.com www.searchlightonline.com www.raybiotech.com www.bdbiosciences.com www.sigmaaldrich.com www.protometrix.com www.molecularstaging.com www.zeptosens.com www.ciphergen.com

Provider Luminex Corporation Bender Medsystems Bio-Rad Laboratories BioSource International INOVA Diagnostics, Inc LINCO Research, Inc. Qiagen Radix BioSolutions R&D Systems Rules Based Medicine Inc. Upstate Biotechnology BD Biosciences

Applications/Kits xMAP , Luminex 100 Fluorescent bead immunoassays (FBIs) Bio-Plex array Multiplex antibody bead kits QUANTA Plex LINCOplex cytokine arrays LiquiChip Customized assay products for research Fluorokine MAP cytokine arrays Species-specific multi-analyte profiles Beadlyte BDcytometric bead array (CBA)

Web link www.luminexcorp.com www.bendermedsystems.com www.bio-rad.com www.biosource.com www.inovadx.com www.lincoresearch.com www.qiagen.com www.radixbiosolutions.com www.rndsystems.com www.rulesbasedmedicine.com www.beadlyte.com www.bdbiosciences.com

BD Biosciences
a

Examples of commercially available miniaturized and parallelized protein assays. Planar protein arrays are offered for reverse screening, cytokine profiling, comparative protein profiling and protein interaction studies. Bead-based systems are mainly offered for focused protein profiling of, for example, cytokines and cell signaling molecules. So far, most of the commercially available bead based protein microarrays are based on the Luminex100 platfom.

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h
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P

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Figure 1. Types of protein interaction microarrays and protein capture microarrays. Specific protein capture on microarrays can be performed using affibodies (a), aptamers (b) or antibodies (c, d) as immobilized specific capture reagents. Detection of bound analytes is usually performed by direct labelling of the analyte molecules (ac) or by antibody sandwich formation (d). Such assays can be used for protein identification and quantification and therefore will be useful tools in multiparametric diagnostics in the near future.The reverse screening approach uses immobilized cell lysates, which represent the whole repertoire of proteins in cells at a distinct state. Unspecific adsorption of protein mixtures can be governed by electrostatic, hydrophobic van der Waals, or metalchelate interactions. Captured proteins can be identified using mass spectrometry (e.g.SELDI) (e) or specific antibodies (f). Different antibodies or patient sera can be used to screen these microarrays for the presence of absence of distinct target proteins (f). Immobilized tissue samples (g) or cells (h) can be used for reverse screening approaches with antibody detection of specific markers as well. Reversed screening assays have a high potential as tools for biomarker detection in proteome analyses. Specific interaction microarrays have been described for receptorligand (e.g. small molecule drug candidates, phospholipids) interactions (i), enzymesubstrate interactions (j), proteinprotein interactions (k), proteinoligosaccharide interactions (l) and proteinDNA interactions (m) and can be used to identify direct 1:1 interaction partners of proteins in a highly parallelized manner.

molecules and protein targets is the first step on the cumbersome path to establishing protein microarrays for proteomics research. We must not forget that proteins must be immobilized without damaging their tertiary structure to retain their functionality. This is, of course, much more difficult than attaching oligonucleotides or PCR fragments onto a microarray slide. However, microarray surfaces have been optimized in a way that keeps proteins in a functional state. In addition, the knowledge on additives protecting protein functionality has also increased and the successful application of protein microarray technology has already been proven [8,9]. In the following sections we will describe examples of different types of protein microarray assays used in protein expression profiling, interaction studies and reverse screening (Fig. 1).

Protein-profiling microarrays

Unfortunately, proteins are not as easy to handle; it is impossible to predict high-affinity capture molecules for proteins on the basis of their primary amino acid sequence alone, due to their most diverse and individual tertiary molecular structures and their innumerable ways of interacting. These interactions are driven by strong electrostatic forces, hydrogen bonds or weak hydrophobic Van der Waals interactions or by all of these in combination. In addition, proteins are not limited to one-to-one interactions but can also interact with different binding partners simultaneously. Hence, they often appear in complexes. Steady or dynamic post-translational modifications such as glycosylation or phosphorylation also have an enormous additional influence on protein interactions. Therefore, each capture molecule must be generated individually following the screening of large sets of candidate capture molecules against their individual target proteins. There are no PCR equivalents available for proteins. Therefore, the development of methods for the cost-effective and fast high-throughput generation of highly-specific, high-affinity protein capture

So far, antibodies have proven to be excellent partners in protein microarrays. However, antibodies be they polyclonal or monoclonal have some disadvantages with regard to their generation and costs. The continuous culture of hybridoma cells, required to produce monoclonal antibodies (mAB), offers an unlimited supply of reagents. By contrast, the supply of polyclonal antibodies is rather limited [1013]. The virtually unlimited supply enables the standardization of both reagent and assay techniques. However, because of the labor-intensive nature of mAB production, further efforts had to be undertaken to develop suitable alternatives. Promising approaches involve phage display techniques [14,15] aptamer technology [16,17] and the use of protein scaffolds such as receptins, trinectins, ankyrins, and anticalins [1821] (Fig. 1a,b). Compared with the highly developed antibody technologies, these alternative approaches are still in their infancies although progress is moving at a fast pace. The potential of specific protein capture microarrays has already been demonstrated [22]. Similar to the dual-colorlabeling approach used for the visualization of differential mRNA expression, studies have been undertaken on the differential display protein analysis. Antibodyantigen

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microarrays were used in which two different protein samples were labeled with two different fluorophores; equal amounts of total protein were mixed and incubated on the antibody microarray (Fig. 2, Fig. 1c) [22]. The differences in the concentrations of the target proteins in each sample on each capture spot were then made visible through dual wavelength fluorescence. Depending on the affinity of the antibody to its antigen, even pM antigen concentrations could be detected [22]. However, one has to be aware that proteins are often assembled into multiprotein complexes. A strong signal can either result from a large amount of captured target protein or from the capture of a huge complex of different proteins. Sreekumar et al. [23] have used antibody microarrays to investigate the changes in the protein expression level of LoVo Colon carcinoma cells after ionizing radiation treatment. Knecevic et al. [24] were able to detect cancer-specific alterations in the expression of proteins with antibody arrays consisting of >100 capture molecules. The two research groups were able to identify more than ten proteins that altered their expression level in response to either ionizing radiation treatment or in correlation to tumor progression. These results were verified using alternative methods such as western blots. Antibody microarrays composed of up to several hundred monoclonal antibodies have become commercially available (Table 1). Belov et al. [25] have used antibody microarrays to determine >50 CD antigens present on leukocytes or leukemia cells. Instead of applying lysates to the array, a cell suspension was incubated onto the array instead. Only those cells expressing the corresponding CD antigen bound to the corresponding antibody microspots. Different patterns of cell binding have been obtained for normal peripheral blood leukocytes and different types of leukemia. This type of antibody microarray can be used to perform immunophenotyping. Whether such assays can compete with the advanced FACS technology still needs to be shown. The peptidomics approach avoids the possibility of capturing large protein complexes on a microspot [26]. The proteins are enzymatically cleaved into peptides which are then captured on arrays of peptide-specific antibodies. The bound peptides are identified by matrix-assisted laser desorption mass spectrometry (MALDI) which circumvents some of the drawbacks of direct protein capture assays such as protein solubilisation, protein complex formation, etc. However, this method still has to show its value for large-scale protein profiling. A more focused protein quantitation approach employs miniaturized and parallelized sandwich immunoassays (Fig. 1d), either using planar-microarray-based systems or bead-based assays (Table 1, Fig. 3). Sandwich immunoassays

Sample A

Sample B

Protein extraction Differential labelling mixing

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Figure 2. Principle of differential-capture protein microarray assays. Proteins from controls and samples are isolated, labelled with two different colors (fluorophores), mixed and incubated on the same protein-capture microarray.The dual-color detection of bound proteins reveals the differences in protein expression between control and sample directly.

have been successfully adapted to the microarray format [2733]. Ekins and colleagues have developed a very sensitive microarray-based analytical technology and were able to prove the high sensitivity of the miniaturized assay. Analytes such as thyroid stimulating hormone (TSH) or HepatitisB surface antigen (HbsAG) could be accurately quantified down to the femtomolar concentration range (corresponding to 106 molecules/ml) [33]. Schweitzer et al. have published the most complex multiplexed sandwich immunoassay so far, which allowed the quantification of 75 different cytokines [32]. However, it was not possible to perform a 75-plex sandwich immunoassay within a single microarray because of cross-reactivity of some of the detection antibodies with immobilized capture antibodies or bound analytes. To separate the different cross-reacting antibodies, two sets of multiplexed sandwich immunoassays were generated, containing 37 or 38 distinct features in a single microarray, respectively. These microarrays were used to study the expression of target protein present in lipolysaccharide or TNF--stimulated and non-stimulated human dendritic cells. The highly sensitive isothermal rolling circle amplification method was used for detection purposes and different cytokines were detected in the femtomolar concentration range. Hence, the rolling circle amplification method has turned out to

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Planar array ID of capture agent: y x = Bead array

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Fluorescence Radioactivity Chemoluminescence etc.

Fluorescence

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Figure 3. Different types of microarrays. Either planar microarrays or bead-based arrays can be employed for multiplexed ligand-binding assays. Planar microarrays can be generated with hundreds and thousands of different capture spots whereas multiplexing in beadbased arrays is limited to the number of distinguishable beads.The separation of beads is performed via colour- (Luminex) or size-coding (BD) of capture beads. In planar arrays analyte spots are easily distinguishable by their xy-coordinates in the array. Detection on planar arrays is performed using either chemoluminescence, radioactivity, mass spectrometry or fluorescence.The latter is the preferred detection method for bound analytes in bead-based microarray assays.

samples such as patient serum or cell culture supernatant [3538]. Several companies offer a steadily growing list of readyto-use multiplexed sandwich immunoassays based on the bead-based Luminex technology to quantify, for example, cytokines, cell signaling molecules, and to analyze kinase activities (Table 1). Rules Based Medicine Inc. (www.rulesbasedmedicine.com) offers a special service which quantifies ~90 mouse serum parameters and ~170 human serum parameters from minute amounts of sample volumes. Using the bead-based technology, thousands of samples can be screened within a very short time. Throughput seems no longer a problem for the bead-based systems and nor does sample volume. Recently, the BD FACSArray Bioanalyzer (www.bdbiosciences.com) was launched as an alternative bead-based system (Cytometric Bead Array, BD CBA). This bead-based system can discriminate different bead sizes and uses a two-color detection, hence allowing the design of more complex assays. Planar microarrays or the bead-based types of miniaturized and multiplexed immunoassays offer an appropriate solution for screening approaches in which the amount of sample is the limiting factor, for example mouse serum samples. They are powerful innovations which help to identify new individual and multiplexed sets of biomarkers related to disease predisposition, progression and response to treatment as well as helping to characterize the patients response to the treatment of certain drug candidates. Therefore, planar microarrays or bead-based assays can become powerful tools for clinical studies.

Microarrays to study interaction and function of proteins

be a feasible way to detect single binding events on microspots [34]. Molecular Staging Inc. (www.molecularstaging. com) uses this technology to offer a service for multiplexed protein analysis designed to yield a comprehensive look at the biology of the underlying disease and drug response. As an alternative to planar microarrays, bead-based systems are of particular interest (Fig. 3), especially when the number of parameters to be determined in parallel is rather low. All bead-based assay systems employ color- or sizecoded microspheres as the solid support for the capture molecules. The beads are identified in a flow cytometer; the amount of captured target proteins is quantified on each individual bead. Sensitivity, reliability and accuracy are similar to those observed with standard ELISA procedures. This technology has been commercially available for a few years and miniaturised multiplexed ligand-binding assays can be performed (xMAP system, Luminex, Austin, TX, USA). The xMAP system has been used to determine the concentration of cytokines or antibodies in biological Protein microarrays are perfectly suited for studying proteinprotein, enzymesubstrate, proteinDNA, protein oligosaccharide and proteindrug interactions simultaneously (Fig.1jm). The first global protein microarray was used to study proteinprotein interactions. Zhu et al. generated a yeast proteome chip containing 5800 recombinant proteins [39]. Binding of calmodulin to the yeast proteome microarray was used to study calmodulinprotein interactions. Binding to known CamKinases and calcineurins was shown and 33 new potential binding partners of calmodulin were identified. In addition, investigations of phospholipidprotein interactions revealed 150 different yeast proteins including integral membrane proteins and periphery-associated proteins. Many of the hitherto uncharacterized proteins are said to be membrane-associated, which was taken as an indication that they preferentially bind specific phospholipids in vivo. This approach is now commercialized by Protometrix Inc (www.protometrix.com) and offers an

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interesting possibility for studying drugprotein interactions or the influence of a given drug in proteinprotein interactions. Enzymesubstrate arrays (Fig. 1j) have been described for different kinds of enzymes including restriction enzymes, peroxidase, phosphatase and protein kinases [4047]. Kinases are one of the protein families with a high potential for drug target candidates. Therefore, the pharmaceutical industry has a strong interest in appropriate assay systems which enable the screening of drugs candidates for kinase activity at a very early stage during drug development. Protein and peptide microarrays are perfectly suited for this approach. Zhu et al. [42] have analyzed the activities of 119 of the 122 known or suspected protein kinases from Saccharomyces cerevisiae for 17 different substrates and came up with hitherto unknown kinase activities. Peptide arrays involving large sets of individual peptides or peptide libraries offer a very interesting alternative to approaches involving recombinant proteins. Pre-synthesized peptides or the on-spot-synthesis of defined peptides have been used to screen for unknown enzymatic activities or antibodies [46,47]. Jerini AG offers their PepStar peptide microarray services to screen for specific substrates of kinases and proteases. Recently, PepScan (www.pepscan.nl) launched their PepChip Kinase microarray allowing one to select the optimal peptide substrate for high throughput assays, to generate a fingerprint of cellular kinase activity, or to analyse the effects of kinase inhibitors within a single experiment. Peptide microarray technology is a powerful technique for proteome analyses enabling studies of molecular recognition events and the identification of biologically active peptides. DNAprotein interactions in a microarray format have been described by Bulyk et al. [41,48] (Fig. 1m). The authors have demonstrated that DNA microarrays can be used to study the activity of restriction enzymes and to characterize sequence-specific DNA recognition by zinc-finger proteins. In general, DNAprotein interaction assays have proven useful in the characterization and identification of DNAbinding proteins. Whether these approaches can be integrated into the drug discovery pipeline remains to be seen. The same is true for glycoarrays, another new type of a microarray (Fig. 1l). Carbohydrates are the key components of glycoproteins, glycolipids and proteoglycans and are involved in recognition processes such as cell adhesion, migration and signaling. Proteincarbohydrate interactions are essential for many biological processes including normal tissue growth and repair, cellcell adhesion and inflammation, cell growth, fertilisation, viral replications and parasitic infection, as well as tumor-cell motility and progression. In addition, alterations of glycosylation events

are also involved in several diseases. Therefore proteincarbohydrate interaction studies based on oligosaccharide microarrays have become valuable research tools [49,50]. We will see in the near future whether these approaches can also be integrated into the drug discovery pipeline. Small molecule arrays (Fig. 1i) might be very interesting applications of microarrays in drug discovery processes. Microarrays generated from a large number of small organic molecules can be used to analyze receptorligand interactions and identify lead structures for drug development. MacBeath et al. [51] have demonstrated the feasibility of such an approach. They synthesized a library of chemical compounds. Each chemical entity was immobilized on a single resin bead which was placed in a distinct well of a 96-well microtiter plate. The organic molecules were chemically released from the beads, diluted, spotted and covalently attached to derivatized glass slides. Incubation of these small-ligand arrays with receptor molecules resulted in binding of the proteins to individual compounds immobilized within distinct microspots. New receptor ligands could thus be identified. This technology enables parallel high-throughput screening for ligandreceptor interactions and only requires very small quantities of valuable samples. This might certainly help improve screening procedures for active substances in pharmaceutical industry. The robustness of these assays has still to be demonstrated in large-scale studies.

Reverse microarrays
An alternative microarray set-up is the reverse screening approach (Fig. 1ef), a method in which tiny amounts of a tissue or cell sample are immobilized in a microarray format onto a solid support [52,53]. Lysates are prepared from differentially treated cultured cells or microdissected tissues, for example procured by LCM (Laser capture microdissection), and arrayed in a microarray format. The immobilized sample proteins thus represent the whole repertoire of proteins in cells in a distinct state. Different antibodies or patient sera can be used to screen these microarrays for the presence or absence of distinct target proteins. This setup allows the screening of a large collection of tissue or cell lysates with a large numbers of antibodies or patient sera. One of the major advantages of reverse microarray screening is that the sample proteins do not need to be labeled or denatured; non-denatured lysates can be used [5355]. The reverse screening approach allows the identification of new biomarkers, the analysis of protein expression profiles and enables early profiling of drug candidates for efficacy and toxicity. Zeptosens AG (www.zeptosens.com) offers a reverse screening platform (CeLyA cell lysate arrays) which is able to detect a defined set of proteins or protein

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modifications of interest using multiplexed, direct affinity assays. Their system enables a much higher efficiency compared to classical western blotting approaches. Ciphergen Biosystems Inc. (www.ciphergen.com) has established another reverse screening technology. The SELDI (Surface Enhanced Laser Desorption Ionization) approach (Fig. 1e) uses mass spectrometry as the read-out system [56,57]. Cell extracts derived from different sources are incubated on different spots of the same adsorptive surface chemistry (e.g. cation/anion exchange material, hydrophobic surfaces). Unbound or weakly bound proteins can be washed away, whereas the whole variety of non-specifically captured target proteins can be analyzed by mass spectrometry. The mass spectrum mirrors the different molecular weights of the captured proteins. The comparison of two MS datasets generated from two different samples immediately identifies the differentially expressed proteins by their molecular mass. In some cases the differentially displayed proteins might be identified immediately on the basis of their molecular weights. However, in most cases it is necessary to identify such markers separately. Analyte proteins are enriched by affinity chromatography, which can easily be achieved using the same adsorptive material as used for the SELDI experiment. The enriched proteins can then be identified by standard methods (e.g. Edman sequencing, western blot, digest mass fingerprinting). The SELDI technology is easy to handle and suitable for the fast detection of differences in total protein content of different samples. As the detector sensitivity of time-of-flight mass analyzers decreases with increasing molecular weights, SELDI is perfectly suited for the detection of small proteins and peptides but exhibits limitations with respect to highmolecular-weight proteins or membrane proteins. Although assay sensitivity in SELDI experiments is much lower in comparison to sandwich immunoassays [58], the SELDI approach still is a quick screening platform for any unknown protein biomarker. Currently, the most prominent reverse screening approach is represented by tissue microarrays (Fig. 1g). This technology was first described by Juha Kononen and Olli Kalloiniemi, who generated a microarray of tissue samples that contained hundreds of tissue specimens [59]. These tissue microarrays were screened for the presence or absence of DNA of RNA molecules or proteins such as p53 or the tumor markers Her-2 and EGFR. Detection was performed by standard analytical methods, such as immunohistochemistry, fluorescence in situ hybridization (FISH), or other molecular detection methods [59,60]. One significant advantage of tissue microarrays compared to conventional histology is the large number of specimens that can be treated simultaneously in an identical manner.

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The conventional histological analysis of tissue specimens is a rather slow and labor-intensive process. The parallel processing of a large number of histological samples dramatically increases the throughput. Therefore, tissue microarrays are powerful research tools to screen a large number of samples for well-defined parameters. However, one disadvantage of tissue microarray analysis remains, especially when regarding them as a diagnostic tool for individual cases. It is difficult to say whether a biopsy is representative for the whole specimen, as it only represents a tiny fraction of the whole tumor.

Outlook
Although the principles of protein microarray technology were already described and established several years ago, its full potential is only now becoming clear. Protein microarrays are powerful tools that can be used for the identification and quantification of proteins and for the analysis of interactions between proteins with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA (Fig. 1). The bottleneck within the field of protein microarray technology is characterized by the limited availability of highly specific and selective capture molecules. Improvements in the generation of large sets of recombinant proteins and high throughput generation of capture molecules will certainly contribute to widening this bottleneck in the near future. Currently the bead-based systems are much further developed with respect to robustness and automation than planar arrays. Today bead-based systems are already on their way to being used in high-throughput applications and routine multiplexed diagnostics. Proteomic research, high-throughput drug compound screening and diagnostic applications will lead to a growing demand for protein microarray technologies. In medical research, protein microarrays can significantly accelerate immune diagnostics by enabling the analysis of all relevant diagnostic parameters simultaneously. In addition, the reduction of sample volume is of great importance, in particular on occasions when the samples are limited as in the analysis of multiple tumor markers from biopsy material. Screening of large well-defined sample collections will allow the identification of panels of biomarkers related to disease predisposition, progression and response to treatment. Microarray technology will help accelerate the drug development process and uncover new possibilities with respect to patient monitoring during clinical tests, disease treatment and therapy.

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