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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
Author(s) -
Yamaguchi Minoru,
Nakayama Daisuke,
Shima Keisuke,
Kuyama Hiroki,
Ando Eiji,
Okamura Takaaki,
Ueyama Norikazu,
Nakazawa Takashi,
Norioka Shigemi,
Nishimura Osamu,
Tsunasawa Susumu
Publication year - 2008
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3735
Subject(s) - chemistry , performic acid , mass spectrometry , tandem mass spectrometry , chromatography , avidin , peptide , amino acid , matrix assisted laser desorption/ionization , tandem mass tag , protein mass spectrometry , sample preparation in mass spectrometry , biotin , biochemistry , combinatorial chemistry , proteomics , desorption , electrospray ionization , organic chemistry , quantitative proteomics , adsorption , gene
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.