SPOT peptide synthesis

Figure 7.5. Peptide array construction by SPOT-synthesis. fl-alanine groups (b-A) interact with the cellulose filter that serves as a planar support. Peptide synthesis then proceeds using Fmoc chemistries using the fl-alanine group as a starting point. The peptide is attached to the filter via its carboxy-terminus. In this case, lysine is added at the second position and various amino acids are present at the amino terminus of the peptide.

The enantiomeric purity of protected amino acids used in peptide synthesis can be determined by pre-column partial deprotection followed by derivatization with Marfey s reagent (116). The Marfey diastereoisomers can be easily resolved and determined by RP-HPLC using an ODS-Hypersil column288. Fifteen amino acids collected from mammalian tissues were derivatized with Marfey s reagent and subjected to two-dimensional TLC. Each individual spot (enantiomeric mixture of a diasteroisomer) was then resolved by RP-HPLC. Except for tyrosine (46) and histidine (117), subnanomole quantities of enantiomers could be analyzed289,290. 

The Spot Technique Synthesis and Screening of Peptide Macroarrays on Cellulose Membranes... 

SPOT synthesis Structure determination not needed convenient on-paper assay moderately expensive spot synthesis equipment and custom-spot peptide membrane are commercially available. Peptide library is relatively small limited to binding and simple functional assay if bound peptides are used for the assay releasable peptides possible, but each in small quantity and peptide spot membrane generally not recyclable for subsequent use. 

Combinatorial libraries are prepared by the (1) parallel synthesis of arrays, (2) split-pool method, (3) biological method, or (4) spatially addressable parallel synthesis [74,78-80]. Parallel synthesis is carried out by the simultaneous synthesis of an array of different compounds. Several methods are available. In the multipin method, the peptide synthesis is carried out on polyethylene rods that have attached protected amino acids [81]. The amino acid sequence of a synthesized peptide on a particular pin depends on the order in which the amino acids are added. The number of products synthesized is the same as the number of pins. Another version of parallel synthesis, known as the teabag method, uses resin-filled bags in place of pins [74]. By pooling the resin portions from the appropriate bags, followed by redistribution and further coupling with a specific amino acid, a peptide library can be synthesized. The SPOT method uses a cellulose paper membrane as a solid support, which acts as an open reactor. Respective reagent solutions are pipetted onto several spots to synthesize as many peptides as the spots chosen [74,82]. 

Multiple peptide synthesis with the SPOT-technique... 

Paper as a solid support fascinated Frank, and he later developed another unique and ingenious technique, the spot synthesis (SPOT). In SPOT synthesis, peptides are synthesized on functionalized membrane sheets as spots that could be as large as 10 mm or as small as 1 mm. The spot synthesis technique became a popular tool for peptide synthesis and was soon automated." The first semiautomated SPOT synthesizer, the ASP222, was launched by ABIMED Analysen-Technik of Germany in 1993." The automated method provided an economical way to synthesize very large numbers of peptides. The Intavis synthesizer Auto-Spot (www.intavis.com) allowed the simultaneous synthesis of 1600 peptides. The chemical and physical problems associated with the use of cellulose sheets as synthesis support were, for the most part, overcome by developing synthetic membranes from polypropylene and Teflon. ... 

The SPOT-synthesis method also employs Fmoc chemistry but uses hydroxyl groups present on cellulose filter paper to derivatize and thereby immobilize (3-alanine groups onto the paper. After deprotection, the 13-alanine groups can be used as platforms for the synthesis of peptide arrays (Fig. 7.5) (Frank, 1992 Gausepohl et al., 1992). This method has been widely used for mapping antigen-antibody interactions as well as protein-DNA, protein-metal and other protein-protein interactions (Reineke et al., 2001). 

The most frequent application of SPOT-synthesis has been in the preparation of peptide arrays for the identification of linear B-cell epitopes. If the protein antigen is known, a set of overlapping peptides that encompass the entire sequence can be readily synthesized and assayed for binding of antibody (Reineke et al., 1999). The individual residues critical for binding can then be determined by SPOT-synthesis of peptides containing amino acid substitutions. 

The PNA chain was linked to the peptide spacer glutamic acid-(y-tert-butyl ester)-(fi-aminohexanoic acid)-(fi-aminohexanoic acid) (Glu [OtBuj-fiAhx-fiAhx) via an enzymatically cleavable Glu-Lys handle. The Glu [OtBuj-fiAhx-fiAhx spacer was coupled to the amino-functionalized membrane by standard Fmoc-Chemistry. Then the membranes were mounted in an ASP 222 Automated SPOT Robot and a grid of the desired format was dispensed at each position. The free amino groups outside the spotted areas were capped and further chain elongation was performed with Fmoc-protected PNA monomers to synthesize the desired PNA oligomers (18). After completion of the synthesis, the PNA oligomers were cleaved from the solid support by incubation with bovine trypsin solution in ammonium bicarbonate at 37 °C for 3 h. 

Oligonucleotide and peptide microarrays can be prepared in situ with light-directed synthesis on a glass surface in conjunction with either a photolithographic method or using a micromirror device (2,11-15). However, these methods are not useful for most organic synthesis. Furthermore, such approaches require equipment that is not readily available. Spot synthesis on cellulose membrane is another in situ synthesis method, but the resulting microarrays are low density (16). 

---