A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis

A procedure is described which provides high yields of pyridylethylated cysteine during gas-phase sequencing of peptides. The method decreases transfer losses by reducing the number of transfer steps required for reduction, alkylation, prior to sequencing a peptide. Proteins bound to polybrene-coated glass-fiber filters used in a gas-phase sequenator may be reduced, pyridylethylated, and sequenced directly on the filter. Reduction of cystine-containing peptides is performed using the nonnucleophilic reductant, tributylphosphine [U. T. Rüegg and J. Rudinger (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 47, pp. 111-116, Academic Press, New York] with concomitant alkylation by 4-vinylpyridine in a buffer soluble in the organic phase. Excess reagents and the buffer are removed by drying the filter and briefly washing with chlorobutane. No N-alkylation is apparent under the conditions described, nor are any amino acid side chains modified. The procedure affords high yields and is particularly useful when subnanomole levels of material must be reduced and alkylated.