Review
Applications of peptide arrays prepared by the SPOT-technology

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Abstract

The growing range of applications for peptide arrays synthesized on coherent membranes by the SPOT-synthesis method proves they have emerged as a powerful proteomics technique to study molecular recognition events and identify biologically active peptides. Several developments, such as the introduction of novel polymeric surfaces, linkers, synthesis/cleavage strategies and detection methods, are facilitating an increasing spectrum of accessible compounds and applications in biological or pharmaceutical research.

Introduction

Published in 1992 by Ronald Frank in Tetrahedron [1], the SPOT-synthesis method opened up countless opportunities to synthesize and subsequently screen large arrays of synthetic peptides on planar cellulose supports. Discrete spots are arranged as arrays on membrane sheets where each spot is individually accessed by manual or automated delivery of the appropriate reagent solutions. Although the SPOT-synthesis method is not as impressively miniaturized as the Affymax system [2], it fulfils similar demands, with the added advantages of relatively simple experimental procedures, inexpensive equipment requirements and highly flexible array and library formatting. Some further advantages of peptide arrays prepared by the SPOT-technique are summarized here. Firstly, this method permits rapid and inexpensive highly parallel synthesis of huge numbers of peptides or peptide mixtures. Secondly, a variety of unnatural building blocks can be used. Thirdly, cellulose or other polymeric membrane supports are compatible with many binding, enzymatic and cellular assays. Finally, the synthesized libraries can be screened in parallel directly on the solid phase without expensive robots. Therefore, peptide arrays prepared by the SPOT-technique became popular tools for studying numerous aspects of molecular recognition. Over the past few years protein–protein recognition, peptide–metal ion interactions, peptide–nucleic acid binding, enzymatic modification of peptides and the direct action of peptides in vivo have all been explored using synthetic peptide arrays on planar supports.

Here we review the latest developments in the generation and application of synthetic peptide arrays. This contribution mainly focuses on progress made in 1999 to 2000, but will also consider some articles of outstanding interest published before 1999.

Section snippets

Synthesis

With respect to the biological assay requirements, the general strategy for assembling peptide arrays by the SPOT-technique is shown in Fig. 1. The methodology has recently been reviewed [3], [4], [5], [6], [7], [8, [9, [10] and detailed synthetic protocols have already been published [3], [7], [8, [10].

The simultaneous generation of peptide arrays by SPOT-synthesis on planar supports exclusively uses the Fmoc peptide synthesis strategy, whereas different kinds of planar supports are currently

B-cell epitope and paratope mapping

The most frequent application of peptide arrays prepared by SPOT-synthesis is the identification of linear peptide epitopes that bind to antibodies (reviewed in [16radical dot]). If protein antigens are known, such screening can be knowledge-based using scans of overlapping peptides derived from the appropriate protein sequences (pepscan). Performing substitutional analyses that comprise all single-site substitution analogues of the wild-type sequence allows easy identification of the residues critical

Protein–protein interactions

Most of the approaches described have been similarly applied to protein interactions in general and recognition motifs of signal transduction domains in particular. These have been studied extensively as most of them bind to short linear peptide motifs. Only a brief summary of some examples will be included here.

One study characterised the proline-rich binding motifs of the EVH1 domains present in proteins of the Ena-VASP family that bind to the mammalian zyxin and vinculin proteins, as well as

Enzyme–substrate recognition

As well as standard binding experiments, cellulose-bound peptide arrays have been successfully used to identify, characterise and optimise enzyme substrates. In principal, all enzymes that modify peptides are amenable to such analyses, but so far only assays for kinases and proteases have been described in detail.

Kinase activity is detected by incubating potential peptide substrates bound to cellulose membranes with the enzyme in the presence of [γ-32P]ATP and subsequent autoradiography (for

T-cell epitope mapping

A prerequisite for T-cell stimulation is the binding of peptides from processed antigens to molecules of the major histocompatibility complex (MHC). The question is which part of an antigen forms a T-cell epitope? To investigate this, the SPOT-method is an ideal synthesis technique to prepare large numbers of potentially relevant peptides. It is, however, necessary to release these peptides from the solid support used during synthesis to allow binding to MHC. So far, mostly MHC class II

Other applications

In addition to the broad fields of application described above, some very specific experiments using peptide arrays have also been described. Reuter et al. [45] identified the type II endonuclease EcoRII DNA-substrate binding site by incubating an EcoRII-derived peptide scan with 32P-labeled oligonucleotides. Technetium-99m binding hexapeptides were selected from a combinatorial library [46]. Combinatorial dipeptide libraries were used to elucidate how reactive the amino-termini and amino acid

Conclusion and future perspectives

Despite having enormous potential in the area of proteomics and drug discovery, not many labs have taken advantage of peptide array technologies. In the pharmaceutical industry, they have been almost completely neglected up until now, for several reasons: the focus was on DNA arrays; peptides were regarded as unimportant in drug discovery processes; the technology was not ripe (automated); and the potential applications were not realised. In the emerging era of proteomics, peptide arrays will

Update

A soon to be published paper [49] describes the synthesis of an array of more than 800 variants of a WW protein domain consisting of 44 amino acids, which was successfully employed for a parallel ligand binding assay.

Acknowledgements

We would like to point out that, although outstanding papers were presented [1], [2], [5], [17], [19], [36], [37], [40], [41], we could only highlight those published since the beginning of 1999.

References and recommended reading

Papers of particular interest, published within the annual period of review,have been highlighted as:

  • radical dot of special interest

  • radical dotradical dot of outstanding interest

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