Role of peptide hydrophilicity on determination of microsequencing efficiency

Abstract

The successful sequencing of short peptides on hydrophobic polyvinylidene difluoride membrane (PVDF-P) has been problematic. In this study the sequencing efficiency of various short synthetic peptides on charged-modified PVDF (PVDF-N) and chemically treated glass-fiber membranes or discs has been examined. These modified membranes provided better repetitive yields or sequencing efficiency than the unmodified PVDF-P. In contrast, there were no significant differences among the resulting initial yields for all the tested membranes, indicating that the modified membranes did not interfere with the coupling/cleaving reactions. Methanol at 1% increased the solubility of phenylisothiocyanate (PITC) in heptane for gas-phase delivery during the coupling reaction, whereas this addition of methanol failed to increase the coupling efficiency. Reduction of chemical background noise by replacing triethylamine (TEA) with diisopropylethylamine (DIPEA) also failed to increase the coupling efficiency. Polybrene strengthened the peptide binding to both PVDF-P and PVDF-N, but increased the amount of carry-over of PTH-amino acid from the current cycle to the next. Nonetheless, hydrophilic peptides had higher sequencing recoveries and repetitive yields than hydrophobic peptides when sequenced on all the tested membranes. This relationship was further verified by testing a synthetic peptide with decreasing hydrophilicity by sequential deletions of 2 amino acid residues from its N-terminus. A decreasing sequencing efficiency was observed, which correlated with the reduced hydrophilicity and peptide length. Similar results were obtained when testing peptide fragments with decreasing hydrophilicity by deletions of amino acids from the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)