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Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa
Published on: September 9, 2013
High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry
Minoru Yamaguchi1, Takashi Nakazawa, Hiroki Kuyama
1Life Science Laboratory, Shimadzu Corporation, Kyoto 604-8511, Japan, Department of Chemistry, Nara Women's University, Nara 630-8506, Japan. yamagu@shimadzu.co.jp
Abstract:
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.
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