A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification

We describe a method for generating multiple small sequences from the N ter minal of peptides in unseparated protein digests by stepwise thioacetylatio n and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "seque nce tags" together with the mass of the parent peptide can be used to ident ify the protein in a database. The sequence ladders are generated without t he use of chain terminators or sample aliquoting and the degradation reagen ts are water soluble so that the chemistry can be carried out on peptides i mmobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing. The entir e procedure can be automated, and we describe a prototype device for the pa rallel analysis of multiple samples, We demonstrate the effectiveness of th is chemical tagging method in a comparison with Edman sequencing, peptide m ass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which cons ists of 57 proteins, We show that chemical tagging is a viable first-pass h igh-throughput identification method to be used prior to an in depth MS/MS analysis.