Spot synthesis: observations and optimizations

Positionally addressable syntheses of peptides on continuous cellulose memb ranes (spot synthesis) have often been reported in detail, but important qu estions dealing with synthesis quality, reproducibility and subsequent bind ing assays have largely been under-emphasized. In this report we have inves tigated some of these problems. The most important results were: (i) the si gnal intensity of ligate binding to cellulose-bound peptides and the affini ty 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 ob served in the spot-center compared to the rim. We recommend a peptide densi ty of 10 nmol/cm(2) as a reasonable starting point for further optimization ; (iii) statistical analysis of binding assay reproducibility with more tha n 15 000 peptides resulted in a mean standard signal deviation of 0.18; and (iv) optimization of side-chain deprotection revealed that a 30-min pretre atment of the cellulose with 90% trifluoroacetic acid followed by the stand ard deprotection protocol resulted in higher purity of the synthesized prod ucts.