The SPOT-synthesis technique: Synthetic peptide arrays on membrane supports—principles and applications

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Abstract

Presented first in 1990 at the 21st European Peptide Symposium in Barcelona, Spain [Frank, R., Güler, S., Krause, S., Lindenmaier, W., 1991. Facile and rapid ‘spot synthesis’ of large numbers of peptides on membrane sheets. In: Giralt, E., Andreu, D. (Eds.) Peptides 1990, Proc. 21st Eur. Peptide Symp. ESCOM, Leiden, p. 151.], the SPOT-synthesis method opened up countless opportunities to synthesise and subsequently screen large numbers of synthetic peptides as well as other organic compounds arrayed on a planar cellulose support [Tetrahedron 48 (1992) 9217]. Already in 1991, a commercial kit for manual SPOT-synthesis became available through Cambridge Research Biochemicals (CRB, UK), and in 1993, a semi-automated SPOT-synthesiser, the ASP222, was launched by ABIMED Analysen-Technik, Germany. Both made the technique available to many research laboratories, even those not experienced in or equipped for chemistry. Although SPOT-synthesis is not as impressively miniaturised as, e.g. the Affymax photolithographic technique [Science 251 (1991) 767], it fulfils similar demands with the advantage of a reliable and easy experimental procedure, inexpensive equipment needs and a highly flexible array and library formatting. The method permits rapid and highly parallel synthesis of huge numbers of peptides and peptide mixtures (pools) including a large variety of unnatural building blocks, as well as a growing range of other organic compounds. Further advantages are related to the easy adaptability to a wide range of assay and screening methods such as binding, enzymatic and cellular assays, which allow in situ screening of chemical libraries due to the special properties of the membrane supports. Therefore, peptide arrays prepared by the SPOT-technique became quite popular tools for studying numerous aspects of molecular recognition, particularly in the field of molecular immunology.

Introduction

The currently very successful paradigm in scientific research applying a systematic empirical search rather than an iterative rational design to solve complex scientific questions relies heavily on technologies that permit for a rapid and comprehensive screening of diverse types of molecular probes. Combinatorial chemical and biological syntheses were the pioneering technologies that paved the way, allowing fundamentally new experimental approaches in molecular biology, immunology and drug discovery research (for a discussion on this topic, see: Gallop et al., 1994, Lebl, 1999). Miniaturisation and automation then became central topics by steadily pushing the number of probes and test samples that can be screened in ever shorter times, markedly reducing the assay dimensions and costs, as well as opening access to the analysis of ever smaller sample sizes ranging from very limited biopsy material to even single cells.

In 1988, Ed Southern disclosed his concept of synthesising partial or complete sets of oligonucleotide probes in a microscaled chequered arrangement onto a planar glass surface to be used as a tool to analyse complex nucleic acid samples of genome-wide origins by multiplexed hybridisation (Southern, 1988). This marked another technological breakthrough: microarray technology. The impact of this technology is now being compared to that of the microelectronics revolution. Other methods rapidly complemented Southern's approach such as the photolithographic synthesis of peptides and later oligonucleotides on glass (Pirrung et al., 1990) and the SPOT-synthesis of peptides on membrane supports (Frank and Güler, 1990). Meanwhile, not only chemical in situ synthesis is utilised to generate arrays of sensor probes, but sophisticated chemical printing instrumentation has been developed to dispense minute volumes of solutions of any type of compound for array at densities up to several thousands per cm2 (Phimister, 1999). Microarray technology is a fast developing field because it enables us to utilise and exploit the enormous amount of information generated by genome and proteome research. With respect to the topic of this special issue of the Journal of Immunological Methods, it should be remembered that most of the current high throughput screening methods were initially invented for systematic studies on molecular recognition events in the immune system based on synthetic peptides. This is also true for SPOT-synthesis.

Section snippets

The technique

The basis concept came from observations made previously with combinatorial oligonucleotide (Frank et al., 1983) and peptide synthesis (Frank and Döring, 1988) on separate, labelled membrane (cellulose) disks, indicating that chemical reactions can proceed to completion, when only enough reagent solution is used as can be taken up by the support material itself. This observation suggested that, e.g. individual amino acid coupling reactions in a multiple parallel synthesis scheme could be

Outlook

At present, an increasing number of organisms are becoming completely known, at least at the level of the genome structure. This immediately opens opportunities to extend those dedicated peptide library strategies to genome-coded protein sequence information for a whole chromosome, genome or inter-genome inspection. Such libraries might cover all combinatorial variants of a bioactive amino acid sequence as displayed by a complete genome or a family of genomes (e.g. the genome-specific patterns

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