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Peptide arrays: from macro to micro Ulf Reimer*†, Ulrich Reineke‡ and Jens Schneider-Mergener*§ Over the past decade of proteome research peptide arrays have become a widespread and powerful tool to study molecular recognition events and to identify biologically active peptides. A variety of applications such as epitope mapping, characterisation of protein–protein interactions, enzyme–substrate or inhibitor interactions, and many more, have been published. Today’s technologies for array production, inspired by DNA chips, have recently turned to the miniaturisation of peptide arrays. These advances open up an expanding spectrum of applications and the information obtained will be well-suited to developing substrates and inhibitors for diagnostic and therapeutic purposes. Addresses *Jerini AG, Invalidenstrasse 130, D-10115 Berlin, Germany † e-mail: [email protected] ‡ e-mail: [email protected] § Institut für Medizinische Immunologie, Campus Charité Mitte, Humboldt-Universität zu Berlin, Schumannstrasse 20-21, D-10117 Berlin, Germany; e-mail: [email protected] Current Opinion in Biotechnology 2002, 13:315–320 0958-1669/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0958-1669(02)00339-7 Abbreviations SPR surface plasmon resonance TASP template-assembled synthetic proteins

amount of active protein critically depends on translation control, assisted folding, trafficking, binding to cofactors and interaction partners, protein turnover, as well as posttranslational modification. For example, changes in expression levels of mRNA by a factor of 10 in different cells are observed and interpreted as significant; however, the actual activity of an enzyme can be altered by a factor of several thousand by a single phosphorylation event. This illustrates the need for methods in the biochemistry toolbox to allow rapid and reliable characterisation of proteins in the context of their cellular status. In principle, classical biochemical methods could answer many of the key questions, but with low throughput. Thus, the unmet need was for high-throughput technologies to elucidate such interactions and activity networks. In general, protein and peptide arrays are able to support this new field of research now called proteomics. The development of automated high-throughput cloning and expression of proteins opened up an impressive number of applications [5], represented, for example, by a protein chip containing the vast majority of yeast proteins [6]. Whereas protein arrays are best suited to investigating which proteins interact with each other, peptide arrays enable one to precisely characterize molecular recognition events at the amino acid level. In addition, information obtained from peptide arrays is excellently suited to the development of substrates and inhibitors for diagnostic or therapeutic purposes, driving drug discovery.

Introduction The technological basis for preparing different types of compound library arrays was established in the early 1990s. Generally, this technology is applicable to many different compound classes, such as DNA, proteins, peptides, carbohydrates, peptidomimetics and small molecules [1–4]. Of these, DNA arrays have progressed to becoming standard research and diagnostic tools, owing to the drive within the life sciences over the past decade to decode and interpret the human genome [1]. As the human and many other genome projects reached completion, it became obvious that the information gained is not sufficient to understand life in general, and that the promise of these projects for healthcare was only partially vindicated. The next focus had to be the gene products. A proteome comprises not only sequence information about all the proteins in a cell, but also their quantity, localisation, activity, modifications, interaction networks, and their regulation. DNA microarray experiments may provide information about the expression level of genes and allow comparison of such data in different cell types or states. Despite this insight into the abundance of DNA or mRNA, however, these levels do not necessarily correlate with the abundance of functional protein molecules. The

Peptide macroarrays In the field of peptide arrays, macroarrays paved the way. Although there are also early reports of developments in preparing high-density peptide arrays using light-directed synthesis of peptides on glass surfaces (see later) [7], such techniques were not refined at the time because of their low practicability. Conversely, the SPOT technology is a very rapid, robust and easy-to-use method for preparing peptide macroarrays, the techniques and their applications having been reviewed extensively [8,9]. Apart from several special applications, these peptide arrays are mainly used for antibody epitope and paratope mapping, mapping of protein–protein interactions in general, and the investigation of enzyme–substrate and enzyme–inhibitor interactions (e.g. kinases, proteases, isomerases and chaperones). Here, we will briefly highlight the most recent and innovative developments. Four novel synthetic strategies and their applications were published recently. First, Hahn et al. [10•] described the characterisation of a linear antibody epitope and a related sequence with an aspartate–glutamate exchange by complete substitutional analyses (i.e. an array comprising all possible single-site substitution analogues). The authors

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prepared cyclic epitope analogues by SPOT synthesis, including not only disulfide cyclisations but also for the first time amide bonds between the N-α group and a glutamic acid sidechain. Another study that opened up novel synthetic approaches described the preparation of 1,3,5-trisubstituted hydantoins on cellulose membranes [11]. The synthesis was based on a modified submonomer peptoid synthesis protocol. To date, peptides or peptidomimetics on arrays prepared by SPOT synthesis are usually assembled in a stepwise manner. For longer peptides this is a laborious and timeconsuming procedure. Therefore, in the third approach, Toepert et al. [12•] synthesised an array of 6859 trisubstituted variants of the human YAP (Yes-associated protein) WW domain (44-mer) by a combination of stepwise synthesis and native chemical ligation. The C-terminal part was prepared by SPOT synthesis and the N-terminal portion was synthesised in advance and ligated to the peptides on the cellulose array. This is one example of how to extend the concept of peptide arrays to arrays of chemically synthesised proteins. Finally, another approach to protein arrays was based on a combination of the TASP concept (template-assembled synthetic proteins) [13] and the SPOT technique [14•]. An array of 96 synthetic antiparallel four-helix bundle proteins was prepared by synthesising 16 different peptides in advance, which were then assembled on a solid-phase-coupled decameric cyclic peptide in a combinatorial manner. This array was used for the de novo identification of mono copper(II) and cobalt(II) proteins by incubation with the respective ions and spectroscopic detection methods. The preparation of four-helix bundle protein arrays was pioneered by the same group and initially used to identify haem proteins with predefined redox potentials [15]. The de novo identification of bioactive peptides or peptide epitopes usually uses combinatorial library arrays with peptide mixtures, where a set of peptides with randomized as well as defined positions is synthesised and screened. Subsequently, the most active combination of defined positions is selected and the remaining randomised positions are deconvoluted. To avoid this time-consuming deconvolution procedure, an array of 5520 randomly generated 15-mer L-peptides was used to identify the epitopes of three monoclonal antibodies recognising linear epitopes [16]. The results showed that a limited library diversity, although far from covering the entire sequence repertoire, can be sufficient to identify bioactive peptides. Characteristically, the initial affinity to the binding partner was low and had to be optimised using complete substitutional analyses. The investigation of enzyme–substrate or enzyme–inhibitor interactions is a major focus for peptide macroarray applications. Particularly interesting, was work characterising the subsite specificity of the Escherichia coli integral membrane

protease OmpT by arrays of internally quenched peptides [17•]. This concept had already been introduced [18], but this was the first time the arrays had been used for large integral membrane enzymes. To achieve this, a long polar polyoxyethylene glycol linker (corresponding to roughly 200 carbon–carbon bonds) had to be introduced to increase the peptide accessibility. A second study meriting mention elucidated the binding specificity of E. coli trigger factor, an enzyme with a chaperone activity and a peptidyl-prolyl-cis/trans isomerase (PPIase) activity [19]. The prolyl-independent binding specificity was characterised with arrays of 2842 peptides derived from protein substrate scans of overlapping peptides. These data were then used to develop an algorithm to predict potential substrate peptides. In addition, the authors could show that prolyl-independent binding of peptide substrates and peptidyl-prolyl isomerisation involve the same binding site. Finally, three recent reviews describing peptide macroarrays in general or specific areas of application provide a good source of more detailed information. Laune et al. [20] summarize the identification of biologically active peptides from the complementarity determining regions of monoclonal antibodies using peptide arrays as the first screening tool. The inventor of the SPOT technology, Ronald Frank, published a review article describing the principles, applications, and history of peptide arrays prepared by the SPOT method [21]. Lastly, the introductory article of the book ‘Peptide Arrays on Membrane Supports – Synthesis and Applications’ contains a comprehensive summary of publications concerning peptide macroarrays [22•].

Peptide microarrays Miniaturisation of arrays is an important issue. Typical macroarrays have densities up to 20 spots per cm2, whereas microarrays on glass slides are produced with densities higher than 200 spots per cm2. A major benefit is the reduced amount of analyte required for an experiment. Moreover, accessibility of the sample peptides is higher at planar surfaces. This is a prerequisite for high signal:noise ratios, especially for enzymatic reactions. Glass surfaces present a material of high production tolerance compared with the natural product cellulose, the standard membrane material for peptide macroarrays. Although the loading of glass surfaces is much lower than on cellulose arrays, this is an advantage for enzymatic assays, but can be a disadvantage for the detection of low-affinity peptide–protein interactions. Only a few examples for peptide microarrays have been published so far, but the promising results and the huge number of potential applications suggest an enormous impact of these technologies in the future [23]. As this is a new technology and biotechnology companies are frequently involved, websites are also used here as references. Production

Similar to the production of DNA microarrays, methods are available for the parallel light-directed synthesis of

Peptide arrays: from macro to micro Reimer, Reineke and Schneider-Mergener

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Figure 1 Assay types applied to peptide microarrays. Parts (a) and (b) show the detection of antibody binding to an immobilised peptide. (a) A fluorescently labelled antibody is used. (b) Detection of binding is mediated by a secondary labelled antibody. (c,d) Show the same experimental set-ups for the detection of interactions between a receptor protein and peptides. (e,f) Depict the assay principle for a kinase activity assay. The kinase phosphorylates the immobilised peptide. Detection involves measuring either incorporated radioactivity when using [γ-32/33P]-ATP or specific binding of a fluorescently labelled phospho-specific antibody.

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peptides on glass surfaces, as first described by Fodor et al. [7]. An easier route for peptide synthesis is the use of photogenerated acids [24,25]. Yet another way is coupling pre-synthesised peptides to the chip. One advantage of this approach is the possibility of using purified peptides, thus avoiding false-positive signals in the assays caused by the synthesis of by-products; it is also possible to generate multiple arrays from a single synthesis. Commercially available functionalised glass surfaces include chips modified with amino, epoxide, aldehyde and sulfhydryl groups, gold or carboxylic acids. Several chemical reactions have been sought to facilitate the chemoselective ligation of unprotected peptides onto surfaces. MacBeath et al. [26] used a Michael addition to couple thiol-containing peptides onto maleimide-derivatised glass surfaces. A disadvantage of this method is its limitation to compounds containing a single cysteine to maintain chemoselectivity. In a following report MacBeath and Schreiber [27•] reported the production of microarrays by firstly activating aminofunctionalised chips with N,N′-disuccinimidyl carbonate and N,N-diisopropylethylamine. In a following step, bovine serum albumin was coupled to the activated chips to form a monolayer. The monolayer was then treated with N,N′-disuccinimidyl carbonate and N,N-diisopropylethylamine for crosslinking of the protein monolayer and to provide anchor groups for the coupling of peptides containing amino groups. Falsey et al. [28••] generated slides coated with glyoxylyl groups and coupled peptides with a linker containing oxy-amino groups forming oximes or terminal thiol groups forming thiazolidine rings. The introduction of stearic acid in the slide production process showed the best results for an optimal shape of the applied

aqueous spots on the slides and low unspecific binding in biochemical assays. In another approach to generate suitable surfaces for biochemical assays, Houseman et al. [29••] coated clean gold slides with a monolayer of glycol and hydroquinone-derivatised alkanethiols. After oxidation of the hydroquinone moiety to benzoquinone, these groups react chemoselectively with peptide-cyclopentadiene conjugates. One interesting feature of the gold substrates generated is that surface plasmon resonance (SPR) spectroscopy can be used for the readout of binding events. By using the methods described here, peptide microarrays composed of 2 to 720 different peptides in three or more copies on one chip have been produced and used in different kinds of assays. The aim of these experiments is to identify either binding or enzymatic modification of the potential substrates. Different types of assays have been used to detect such events on peptide microarrays (Figure 1). Binding assays

Binding of macromolecules can be detected using a fluorescently labelled ligand, as described for the detection of streptavidin or avidin–peptide interactions (Figure 1c) [28••]. Here, two peptides, as well as biotin, were presented on the microarrays, the peptides being specific for either avidin or streptavidin. As expected for the binding of Cy3labelled streptavidin to the streptavidin-specific peptide WSHPQFEK (in single-letter amino acid code), a positive (red) signal was observed, whereas the Cy5-labelled avidin resulted in a green signal when binding to the avidinspecific ligand HPYPP. Both ligands bound equally well to

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Figure 2 (a)

Peptide microarrays displaying 720 peptides. The 720 peptides are derived from phosphorylation sites from human proteins that are annotated in the databases SWISS-PROT or Phosphobase [31,32]. Phosphorylation was carried out using the kinase c-Abl and either (a) ATP or (b) [γ-32P]ATP. Phosphorylation was detected by either (a) incubating the chip with a fluorescently labelled phosphotyrosine-specific antibody or (b) autoradiography.

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biotin, leading to the mixed green and red dye signal, yellow. In additional experiments it was shown that the anti-insulin monoclonal antibody (HB125) binds to an immobilised mimotope. This was detected using a fluorescently labelled secondary antibody (Figure 1b). To facilitate label-free detection, a gold chip was tested as a solid support [29••]. This chip was coated with an alkanethiol monolayer and a library of different peptides immobilised to it. After incubation of these microarrays, SPR was used for detection, revealing specific binding of a phosphotyrosine-specific antibody to a phosphopeptide contained in the library. All the assays described here showed no non-specific analyte binding, neither to the surface nor to the control peptides. Enzyme activity

The first experiments for measuring enzyme activity on peptide microarrays were carried out on chips coated with a bovine serum albumin monolayer to which three different specific kinase substrates were linked [27•]. The kinase reaction was carried out in the presence of [γ-33P]-ATP and detected by subsequent autoradiography. Using an immobilised peptide substrate for the protein tyrosine kinase p60c-src, Falsey et al. [28••] showed incorporation of 33P into the peptide (Figures 1e,f). Houseman et al. [29••] used three peptides, one also a known substrate for p60c-src, for assays with this kinase. Specific phosphorylation of the substrate, but not of the control peptides, could be shown using either a phosphotyrosine specific antibody, SPR or incorporation of radiolabelled phosphate. In another experiment, peptide microarrays with an attached p60c-src substrate were used to determine inhibitory constants for two different inhibitors of p60c-src. To prevent evaporation within the reaction time the slides were overlayed with mineral oil. Samples with kinase, [γ-32P]-ATP and different concentrations of inhibitors were pipetted on the slides. After the reaction and

washing, the incorporated radioactivity was measured allowing calculation of the Ki values. By comparison of the data obtained from competitive and non-competitive inhibitors it was obvious that the Ki values could be determined directly, as there is no competition between the substrate and the inhibitor in the assay solution. Peptide microarray technology has also been used to characterise kinase substrate specificity [30]. Two general strategies were applied. In the first approach, peptide scans covering common kinase substrate proteins such as myelin basic protein were printed onto chips. In the second approach, libraries of peptides were used that either contained random sequences or were derived from annotated phosphorylation sites in human proteins [31,32] (Figure 2). Detection of substrates was carried out by autoradiography or binding of fluorescently labelled antibodies. These arrays contained the highest number of different peptides to date, made possible through the use of SPOT technology for high-throughput synthesis of peptide libraries for the microarray production process. This approach was used in a recent study to investigate the substrate specificity of NEK6, a kinase that phosphorylates the hydrophobic motif of S6K and SGK in vivo [33•]. For a more detailed characterisation of substrate specificity a substitutional analysis, of one of the substrates identified, was used in an array experiment where all the substrate residues where substituted by each of the 20 proteinogenic amino acids. Cell adhesion assay

The peptide arrays prepared by Falsey et al. [28••], discussed above, were used for a proof-of-concept study in a cell adhesion assay. Different peptides were immobilised at the glass surface, including a peptide that binds specifically to the surface idiotype of WEHI-231 cells. Adhesion experiments showed that only the targeted cell line bound to the peptides and, moreover, could induce tyrosine phosphorylation as confirmed by a phosphotyrosine-specific antibody interaction.

Peptide arrays: from macro to micro Reimer, Reineke and Schneider-Mergener

Conclusions and future perspectives Over the past decade peptide macroarrays have become a standard tool for studying molecular interactions and have been used in a great variety of applications. Taking advantage of novel production technologies in the field of DNA arrays, developments are turning to the miniaturisation of peptide arrays. Although, so far, only a few studies have been published using peptide microarrays, the outstanding results obtained using higher density microarrays imply that this technology will have a huge impact on key developments in proteomics and drug discovery in the near future.

Update In a recent article, Melnyk et al. [34] report the generation of peptide microarrays by immobilising glyoxylyl peptides onto glass slides covered by a semicarbazide layer. The arrays were used to exemplify the specific detection of antibodies from infected individuals’ blood samples.

Acknowledgements We thank Mike Schutkowski for fruitful discussions and critical reading of the manuscript. We are grateful to Liying Dong for performing the experiment shown in Figure 2.

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10. Hahn R, Welfle H, Wessner H, Zahn G, Scholz C, Seifert M, • Harkins R, Schneider-Mergener J, Höhne W: Cross-reactive binding α antibody Fab fragment: an X-ray of cyclic peptides to an anti-TGFα structural and thermodynamic analysis. J Mol Biol 2001, 314:293-309. This publication describes the conformational stabilization of a peptidic antibody epitope by cyclic analogues including disulfide and N terminus to sidechain cyclisation methods. 11. Heine N, Germeroth L, Schneider-Mergener J, Wenschuh H: A modular approach to the spot synthesis of 1,2,5-trisubstituted hydantoins on cellulose membranes. Tetrahedron Lett 2001, 42:227-230.

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12. Toepert F, Knaute T, Guffler S, Schneider-Mergener J: Combining • SPOT synthesis and native chemical ligation to generate large arrays of small protein domains. In Peptides, The Wave of the Future. Proceedings of the 2nd International and the 17th American Peptide Symposium. Edited by Lebl M, Houghten RA. Dordrecht: Kluwer Academic Publishers; 2001:212-213. The combination of SPOT synthesis and native chemical ligation enables the rapid synthesis of large arrays of small proteins or protein modules. 13. Mutter M, Tuchscherer G: Non-native architectures in protein design and mimicry. Cell Mol Life Sci 1997, 53:851-863. 14. Schnepf R, Hörth P, Bill E, Wieghardt K, Hildebrandt P, Haehnel W: • De novo design and characterization of copper centers in synthetic four-helix bundle proteins. J Am Chem Soc 2002, 123:2186-2195. An elegant combination of the TASP concept and the SPOT method to synthesise protein arrays. 15. Rau HK, DeJonge N, Haehnel W: Combinatorial synthesis of four-helix bundle hemoproteins for tuning cofactor properties. Angew Chem Int Ed Engl 2001, 112:256-259. 16. Reineke U, Ivascu C, Schlief M, Landgraf C, Gericke S, Zahn G, Herzel H, Volkmer-Engert R, Schneider-Mergener J: Identification of distinct antibody epitopes and mimotopes from a peptide array of 5520 randomly generated sequences. J Immunol Methods 2002, in press. 17. •

Dekker N, Cox RC, Kramer A, Egmond MR: Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries. Biochemistry 2001, 40:1694-1701. This paper is noteworthy as arrays of internally quenched peptides are used for the first time to characterise the subsite specificity of a large integral membrane enzyme. 18. Reineke U, Bhargava S, Schutkowski M, Landgraf C, Germeroth L, Fischer G, Schneider-Mergener J: Spatial addressable fluorescence-quenched peptide libraries for the identification and characterization of protease substrates. In Peptides 1998 Proceedings of the 25th European Peptide Symposium. Akadémiai Kiadó: Budapest, 562-563. 19. Patzelt H, Rüdiger S, Brehmer D, Kramer G, Vorderwülbecke S, Schaffitzel E, Waitz A, Hesterkamp T, Dong L, Schneider-Mergener J et al.: Binding specificity of Escherichia coli trigger factor. Proc Natl Acad Sci USA 2001, 98:14244-14249. 20. Laune D, Molina F, Ferrières G, Villard S, Bès C, Rieunier F, Chardès T, Granier C: Application of the SPOT method to the identification of peptides and amino acids from the antibody paratope that contributes to antibody binding. J Immunol Methods 2002: in press. 21. Frank R: The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports — principles and applications. J Immunol Methods 2002, in press. 22. Frank R, Schneider-Mergener J: SPOT-synthesis: scope and • applications. In Peptide Arrays on Membrane Supports — Synthesis and Applications. Springer Laboratory Manual. Edited by Koch J, Mahler M. 2002: 1-22. A comprehensive collection of information for peptide macroarrays. 23. BioInsights: Protein Biochips: on the Threshold of Success. USA: BioInsights; 2000. URL: www.bioinsights.net 24. Pellois JP, Wang W, Gao X: Peptide synthesis based on t-Boc chemistry and solution photogenerated acids. J Comb Chem 2000, 2:355-360. 25. LeProust E, Pellois JP, Yu P, Zhang H, Gao X, Srivannavit O, Gulari E, Zhou X: Digital light-directed synthesis. A microarray platform that permits rapid reaction optimization on a combinatorial basis. J Comb Chem 2000, 2:349-354. 26. MacBeath G, Koehler AN, Schreiber SL: Printing small molecules as microarrays and detecting protein–ligand interactions en masse. J Am Chem Soc 1999, 121:7967-7968. 27. •

MacBeath G, Schreiber SL: Printing proteins as microarrays for high-throughput function determination. Science 2000, 289:1760-1763. First description of diverse applications of peptide and protein arrays. 28. Falsey JR, Renil M, Park S, Li S, Lam KS: Peptide and small •• molecule microarray for high throughput cell adhesion and functional assays. Bioconjugate Chem 2001, 12:346-353. The first report of cell-based assays on peptide microarrays.

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29. Houseman BT, Huh JH, Kron SJ, Mrksich M: Peptide chips for the •• quantitative evaluation of protein kinase activity. Nat Biotechnol 2002, 20:270-274. Use of a label-free method for investigating protein–peptide interactions on microarrays. 30. Jerini on the World Wide Web. URL: http://www.jerini.com/array 31. Kreegipuu A, Blom N, Brunak S: PhosphoBase, a database of phosphorylation sites: release 2.0. Nucleic Acids Res 1999, 27:237-239. 32. Bairoch A, Apweiler R: The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res 2000, 28:45-48.

33. Lizcano JM, Deak M, Morrice N, Kieloch A, Hastie CJ, Dong L, • Schutkowski M, Reimer U, Alessi DR: Molecular basis for the substrate specificity of NEK6: evidence that it does not phosphorylate the hydrophobic motif of S6K and SGK in vivo. J Biol Chem 2002: in press. This is the first practical application of peptide microarrays for the characterisation of kinase substrate specificity. 34. Melnyk O, Duburcq X, Olivier C, Urbes F, Auriault C, Gras-Masse H: Peptide arrays for highly sensitive and specific antibody-binding fluorescence assays. Bioconj Chem 2002; ASAP article.