Automated Edman degradation for sequencing is the classic tool for determining the N-terminal sequences of proteins. Although it has been complemented or largely replaced by a variety of mass spectrometric methods in recent years, there is no simple mass spectrometric method for identification and de novo determination of the N-terminal sequence of a protein. Here, we present a simple and reliable approach which identifies N-terminal peptides of proteins by selectively labeling the N-terminal á-amino groups with a fluorescent reagent followed by enzyme digestion and tandem mass spectrometric analysis using an LC-MS/MS system coupled with a fluorescence detector. This approach also includes a differential peptide mapping step for identification of pyroglutamate-blocked N-terminal peptides. In addition, the strategy can be applied to proteins in gel. With this approach, complete N-terminal sequences of proteins can be obtained precisely and efficiently.
Amino Acid Sequence Chromatography, Liquid