Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion

Peptide mass fingerprinting is a powerful tool for the identification of pr oteins, Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analy zed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation, About 90% of the matched peptides with missed cleavage s ites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines /arginines, and (iii) lysines and arginines with an aspartic acid or glutam ic acid residue on either the amino- or carboxyterminal side. The first gro up is already accounted for by search programs. The number of missed cleava ge sites can be increased without reducing the precision of the database se arch by taking the other two groups into consideration. Peptides with trypt ophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized fo rms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine we re found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine, These data can be used for the refinement of protein searches by peptide mass fingerprinting, Copyright (C) 2000 John Wiley & Sons, Ltd.