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(alpha)-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.
Yamaguchi
Minoru
M
Life Science Business Department, Shimadzu Corporation, Kyoto 604-8511, Japan. yamagu@shimadzu.co.jp
Nakayama
Daisuke
D
Shima
Keisuke
K
Kuyama
Hiroki
H
Ando
Eiji
E
Okamura
Taka-Aki
TA
Ueyama
Norikazu
N
Nakazawa
Takashi
T
Norioka
Shigemi
S
Nishimura
Osamu
O
Tsunasawa
Susumu
S
D000596:Amino Acids / Q000737:chemistry*; D045744:Cell Line, Tumor; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007202:Indicators and Reagents; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins