Peptide arrays Spot synthesis

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. 

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.

Frank, R. (2002) The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports—principles and applications. J. Immunol. Methods 267, 13-26. 

Key Words Spot synthesis peptide array screening detection probing HRP POD biotin-labeling peptide-protein interaction. 

Frank, R. and Schneider-Mergener, J. (2002) SPOT synthesis—scope of applications. In Peptide Arrays on Membrane Support, eds. J. Koch and M. Mahler, pp 1-22. Berlin Heidelberg Springer-Verlag. 

Hilpert, K., Winkler, D.F.H., and Hancock, R.E.W. (2007) 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. Nature Protocols 2, 1333-1349. 

Frank, R. and Overwin, H. (1996) SPOT synthesis. Epitope analysis with arrays of synthetic peptides prepared on cellulose membranes. In Epitope Mapping Protocols, ed. G.E. Morris, pp. 149-169. Totowa, NJ Humana Press. 

A peptide library can be synthesized using the SPOT synthesis technique to form a low-density peptide spot array (e.g., 25 spots/cm ). In this method, different peptides are synthesized in situ as low-density arrays on cellulose membrane or paper (8). The volume of Fmoc-amino acids and coupling reagents dispensed creates a specific SPOT size that determines both the scale of reaction and the absolute number of peptides that can be arranged on an area of a membrane. Cotton (another form of cellulose) and polystyrene-grafted polyethylene film segments also have been used as solid supports. Recently, polymeric membranes that are chemically, mechanically, and thermally more stable have been developed, which include hydroxy-functionalized PEG acrylate polypropylene membranes and an amino-functionalized ester-free PEG... 

Peptide arrays on paper or cellulose membranes generated by SPOT synthesis generally are low density, even with the commercially available automatic SPOT synthesizer. Such a low-density peptide chip (several thousand peptides) now is available commercially, for example, PepChip microarray from Mimotopes. Foder et al. (4) first described the... 

The spot synthesis technique is simple and easily accessible for practical use. There are some limitations to the scope of chemistries that can be used with cellulose as a solid support. To eliminate some of these shortcomings, Gao and EsnoufP° 4 proposed using a different membrane, Immobilon AV-1, for spot synthesis. Before the synthesis of peptide arrays can be carried out, this polyvinylidene fluoride based material is derivatized with ethane-1,2-diamine, followed by coupling of Fmoc-(3Ala as a spacer. In most other aspects, this peptide array synthesis is very similar to the spot synthesis proposed by Frank.t l... 

A different way to produce chemical microarrays in situ is spot synthesis of combinatorial libraries on cellulose sheets [56]. Spot synthesis is configured as an open system to be operated at room temperature. Despite attempts to replace cellulose with polypropylene as a synthesis support [57], cellulose is still the support of choice for spot synthesis, and reaction conditions have to be compatible with the restricted chemical stability of cellulose. Due to the acid labihty of such membranes, the diversity content of these arrays was initially restricted to the synthesis of peptides. Recently, a method was described that could widen the scope of spot synthesis arrays. Germeroth and coworkers [57] succeeded in the assembly of a library of 8000 cellulose-bound 1,3,5-triazines under mild reaction conditions. They employed a strategy that took advantage of a temperature-dependent, successive displacement of cyanuric chlorides by different nucleophiles in a first report of the synthesis of smaU organic compounds on ceUulose sheets. 

Toepert F, Knaute T, Guffler S (2003). Combining SPOT synthesis and native peptide ligation to create large arrays of WW protein domains. Angewandte Chemie Internal. Ed. 42 1136-1140. 

Many of the techniques developed for the production of large arrays of peptides by parallel synthesis, such as T-bag, SPOT, and PIN synthesis, have naturally been included. Finally, a survey of available automated instrumentation has also been provided. 

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