High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry.
A high-throughput method for sequencing of N termini of proteins by using postsource decay (PSD) of matrix-assisted laser desorption/ionization mass spectrometry has been developed. After a protein blotted on the PVDF membrane was successively reduced, S-alkylated, and guanidinated, its N-amino group was coupled to biotinylcysteic acid. The protein was then extracted from the membrane and digested with trypsin. The derivatized N-terminal fragment was then specifically isolated from the tryptic digest with avidin resins, and its de novo sequencing was successfully performed by PSD utilizing a sulfonic acid group introduced to the N terminus.
Yamaguchi
Minoru
M
Life Science Laboratory, Shimadzu Corporation, Kyoto 604-8511, Japan, Department of Chemistry, Nara Women's University, Nara 630-8506, Japan. yamagu@shimadzu.co.jp
Nakazawa
Takashi
T
Kuyama
Hiroki
H
Obama
Takashi
T
Ando
Eiji
E
Okamura
Taka-aki
TA
Ueyama
Norikazu
N
Norioka
Shigemi
S
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D003544:Cysteic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007797:Laminin / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D007797:Laminin; D009842:Oligopeptides; D011506:Proteins; C516561:biotinylcysteic acid; D012710:Serum Albumin, Bovine; D001710:Biotin; D003544:Cysteic Acid; D009113:Muramidase