Identification of cysteine-containing peptides in protein digests by high-performance liquid chromatography.

A method is described for the facile identification of half-cystine residues in peptides from protein digests. Combined use of S-beta-(4-pyridylethylation) of cysteine residues with 4-vinyl pyridine and peptide mapping by reversed-phase high-performance liquid chromatography provides the basis for this procedure. Essentially simultaneous, in-line effluent monitoring at 220, 280, and 254 nm provides highly sensitive, specific, and instantaneous identification of all peptide peaks, as well as those containing tryptophan and half-cystine residues, respectively. The resultant cysteine-derivatized peptides are well suited to amino acid compositional and protein sequence analysis, and are protected from loss during enzymatic digestion and/or peptide mapping.