Identification of phosphorylated and glycosylated sites in peptides by chemically targeted proteolysis.

A chemical strategy has been developed for identifying phosphorylated and glycosylated sites in peptides. Phosphoserine, phosphothreonine, O-glycosylserine, and O-glycosylthreonine residues in the peptides were converted to the protease-sensitive S-2-aminoethylcysteine derivatives by beta-elimination followed by Michael addition of 2-aminoethanethiol.The resultant lysine analogs were cleaved with Achromobacter lysine endopeptidase.The predicted proteolytic fragments were analyzed by mass spectrometry and N-terminal Edman degradation. When acetylation was carried out as a first step, direct N-terminal chemical sequencing of the digests yielded sequences immediately C-terminal to the phosphorylated or glycosylated residues. Hence, assignment of the sites of modification could be obtained from the chemical sequence data or mass data.Thus, the method offers an approach for rapidly sequencing large,multiply phosphorylated and glycosylated peptides derived from post-translationally modified proteins by both mass spectrometry and Edman chemical degradation.