Figure 1: Fidelity and stepwise yield of in situ peptide synthesis.

(a) Cognate epitopes for monoclonal antibodies p53Ab8 (epitope: SDLWKL), p53Ab1 (RHSVV) and DM1A (AALEKD) were synthesized 2,163 times as part of the 330,000 feature array. Average binding levels of each monoclonal antibody (Ab probe) with each epitope (nine measurements) are shown and normalized to binding to the cognate epitope. (b) The DM1A epitope and several variants were synthesized in 200-μm features on the same wafer. Both a MALDI image (different colours represent different molecular weights) and a fluorescence image of labelled DM1A Ab binding are shown. MALDI spectra extracted from several of the imaged features over a 160 D mass range are provided. The small peak to the right of the main peak represents incomplete side chain deprotection. (c) MALDI mass spectra from individual 200-μm features in which peptides containing 21 amino acids plus a tris (2,4,6-trimethoxyphenyl)phosphonium-acetyl (TMPP-Ac) group and a 30-atom polyethylene glycol linker were synthesized. (d) A series of 10-mer peptides were synthesized in 200-μm features that differed only in three amino acids (bold and underlined). The full-length peptide and the expected mass for each of the single deletion products of the variable amino acids are shown (marked by arrows). (e) The yields for each amino acid determined from measurements described in part (d). Measurements of multiple peptides containing each amino acid gave s.e.’s of 1% or less. The conditions used to release the peptides from the surface appear to affect the MALDI signals for Trp and His, so yields for these amino acids are not known.