A Method for the Chemical Generation of N-Terminal Peptide Sequence Tags for Rapid Protein Identification

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• 作者：Sjouke Hoving ; Martin Mü ; nchbach ; Holger Schmid ; Luca Signor ; Anton Lehmann ; Werner Staudenmann ; Manfredo Quadroni ; and Peter James
• 刊名：Analytical Chemistry
• 出版年：2000
• 出版时间：March 1, 2000
• 年：2000
• 卷：72
• 期：5
• 页码：1006 - 1014
• 全文大小：200K
• 年卷期：v.72,no.5(March 1, 2000)
• ISSN：1520-6882

文摘

We describe a method for generating multiple smallsequences from the N terminal of peptides in unseparatedprotein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminallydegraded peptides give short sequences of defined length.Such short "sequence tags" together with the mass of theparent peptide can be used to identify the protein in adatabase. The sequence ladders are generated without theuse of chain terminators or sample aliquoting and thedegradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18reversed-phase supports without any peptide loss due towashing with organic solvents as occurs in Edman typesequencing. The entire procedure can be automated, andwe describe a prototype device for the parallel analysis ofmultiple samples. We demonstrate the effectiveness ofthis chemical tagging method in a comparison with Edmansequencing, peptide mass fingerprinting, and MS/MSanalysis of crude protein fractions obtained from an HPLCseparation of the Escherichia coli ribosome complexwhich consists of 57 proteins. We show that chemicaltagging is a viable first-pass high-throughput identificationmethod to be used prior to an in depth MS/MS analysis.