Spot synthesis: observations and optimizations

Abstract

Positionally addressable syntheses of peptides on continuous cellulose membranes (spot synthesis) have often been reported in detail, but important questions dealing with synthesis quality, reproducibility and subsequent binding assays have largely been under-emphasized. In this report we have investigated some of these problems. The most important results were: (i) the signal intensity of ligate binding to cellulose-bound peptides and the affinity of the corresponding soluble peptides show good correlation, illustrated by three different ligate binding assays; (ii) reducing peptide density on the cellulose avoids the 'ring spot' effect, i.e. where less binding is observed in the spot-center compared to the rim. We recommend a peptide density of 10 nmol/cm2 as a reasonable starting point for further optimization; (iii) statistical analysis of binding assay reproducibility with more than 15000 peptides resulted in a mean standard signal deviation of 0.18; and (iv) optimization of side-chain deprotection revealed that a 30-min pretreatment of the cellulose with 90% trifluoroacetic acid followed by the standard deprotection protocol resulted in higher purity of the synthesized products.

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

“…In enzymatic reactions or protein binding studies it is often observed that the periphery of the peptide spot has more signal intensity than the center ("ring spot effect"). This effect can be due to the high density of peptides in the central part of the spot and then it can be resolved by downscaling the peptide synthesis 22 . For further trouble shooting of peptide synthesis the machine handbook and company service should be consulted.…”

Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose using peptide array technology

“…The limitations of reliability of SPOT technology have been described by several authors [18][19][20] . Since purity and quantity of the peptides synthesized on cellulose may vary, all results achieved using this method need to be confirmed with peptides produced by conventional solid-phase peptide synthesis on resin.…”

Synthetic peptide arrays for investigating protein interaction domains

“…The quality of SPOT-synthesized peptides has been investigated by several groups. Takahashi and co-workers [60] reported peptide purity higher than 92%, while Kramer and co-workers [61] reported lower purities. An extended HPLC analysis showed that purities of SPOT-synthesized short peptides of up to 15 amino acids are similar to those synthesized by solid-phase methods in reactors [62].…”