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...

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Published in:Rapid communications in mass spectrometry 2008-10, Vol.22 (20), p.3313-3319
Main Authors: Yamaguchi, Minoru, Nakayama, Daisuke, Shima, Keisuke, Kuyama, Hiroki, Ando, Eiji, Okamura, Taka-aki, Ueyama, Norikazu, Nakazawa, Takashi, Norioka, Shigemi, Nishimura, Osamu, Tsunasawa, Susumu
Format: Article
Language:English
Subjects:
Amino Acids - chemistry
Cell Line, Tumor
Databases, Protein
Electrophoresis, Polyacrylamide Gel
Humans
Indicators and Reagents
Peptides - chemistry
Proteins - chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Summary: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 Nα‐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.
ISSN:0951-4198
1097-0231
DOI:10.1002/rcm.3735