1. Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion
Kai Hilpert, Dirk F H Winkler, Robert E W Hancock Nat Protoc. 2007;2(6):1333-49. doi: 10.1038/nprot.2007.160.
Peptide synthesis on cellulose using SPOT technology allows the parallel synthesis of large numbers of addressable peptides in small amounts. In addition, the cost per peptide is less than 1% of peptides synthesized conventionally on resin. The SPOT method follows standard fluorenyl-methoxy-carbonyl chemistry on conventional cellulose sheets, and can utilize more than 600 different building blocks. The procedure involves three phases: preparation of the cellulose membrane, stepwise coupling of the amino acids and cleavage of the side-chain protection groups. If necessary, peptides can be cleaved from the membrane for assays performed using soluble peptides. These features make this method an excellent tool for screening large numbers of peptides for many different purposes. Potential applications range from simple binding assays, to more sophisticated enzyme assays and studies with living microbes or cells. The time required to complete the protocol depends on the number and length of the peptides. For example, 400 9-mer peptides can be synthesized within 6 days.
2. Evaluating the coupling efficiency of phosphorylated amino acids for SPOT synthesis
Victor Tapia, Bernhard Ay, Julia Triebus, Eike Wolter, Prisca Boisguerin, Rudolf Volkmer J Pept Sci. 2008 Dec;14(12):1309-14. doi: 10.1002/psc.1068.
A high demand of interest concerning binding assays to study the consequences of posttranscriptional phosphorylation may be addressed by peptide array-based methods. A crucial factor for de novo chemical approaches to generate such arrays is the possibility to rationally permutate phosphorylation events along a huge number of sequences. The simple principle behind this advantage is the stepwise synthesis of peptides, which allows the incorporation of either phosphorylated or nonphosphorylated derivates at serine, threonine, and tyrosine positions. In spite of several reported applications of phosphopeptide arrays, there is, to our best knowledge, no reported analysis of the efficiency of the involved techniques. Here, we analyze different coupling conditions to introduce phosphoamino acids in standard SPOT synthesis. Our results clearly indicate that EEDQ is the preferable activator and can also be used in fully automated SPOT synthesis.
3. The spot technique: synthesis and screening of peptide macroarrays on cellulose membranes
Dirk F H Winkler, William D Campbell Methods Mol Biol. 2008;494:47-70. doi: 10.1007/978-1-59745-419-3_4.
Peptide arrays are a widely used tool for drug development. For peptide-based drug design it is necessary to screen a large number of peptides. However, there are often difficulties with this approach. Most common peptide synthesis techniques are able to simultaneously synthesize only up to a few hundred single peptides. Spot synthesis is a positionally addressable, multiple synthesis technique offering the possibility of synthesizing and screening up to 10,000 peptides or peptide mixtures on cellulose or other membrane surfaces. In this chapter we present the basic procedures and screening methods related to spot synthesis and outline protocols for easy-to-use detection methods on these peptide arrays.
