Selective isolation of N‐terminal peptides from proteins and their de novo sequencing by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry without regard to unblocking or blocking of N‐terminal amino acids

We have developed a new method to determine the N‐terminal amino acid sequences of proteins, regardless of whether their N‐termini are modified. This method consists of the following five steps: (1) reduction, S‐alkylation and guanidination for targeted proteins; (2) coupling of sulfo‐NHS‐SS‐biotin to N α ‐amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease followed by oxidation with performic acid; (4) specific isolation of N‐terminal peptides from digests using DITC resins; (5) de novo sequence analysis of the N‐terminal peptides by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) using the CAF (chemically assisted fragmentation) method or tandem mass spectrometric (MS/MS) analysis according to unblocked or blocked peptides, respectively. By employing DITC resins instead of avidin resins used in our previous method (Yamaguchi et al ., Rapid Commun. Mass Spectrom. 2007; 21: 3329), it has been possible to isolate selectively N‐terminal peptides from proteins regardless of modification of N‐terminal amino acids. Here we propose a universal method for N‐terminal sequence analysis of proteins. Copyright © 2008 John Wiley & Sons, Ltd.