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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


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.

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Figure 1: Preparation of the cellulose membrane.
Figure 2: Amino acid coupling cycle.
Figure 3: Modifications and final deprotection of the peptide.
Figure 4: Dependency of the spot size from the pipetted volume of DMSO on different filter papers ( Whatman 540; ▪ Whatman 50; Whatman Chr).
Figure 5: Picture of a cellulose sheet where 160 peptides were synthesized.
Figure 6: List of 13 different peptide sequences.
Figure 7: A typical HPLC profile of a crude 9-mer peptide with the D-amino-acid sequence NH2–mdeaffd–A′-A′-CONH2 containing two β-Ala (A′) as a spacer.
Figure 8: A MALDI-ToF-MS image of a biotinylated and cystein-cyclized crude 10-mer peptide with the sequence biotin-CSHFNDYC-A′A′-CONH2 containing two β-Ala (A′) as a spacer (calc. MW = 1,353.47).

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We are grateful for financial assistance from the Applied Food and Materials Network and the Canadian Institutes of Health Research. R.E.W.H. was supported by a Canada Research Chair award. K.H. was supported by a fellowship from the Canadian Institutes of Health Research. We thank R.A. Klady for the critical proofreading of the manuscript.

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Hilpert, K., Winkler, D. & Hancock, R. 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. Nat Protoc 2, 1333–1349 (2007).

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