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Chemical derivatization of peptides containing phosphorylated serine/threonine for efficient ionization and quantification in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Author(s) -
Tsumoto Hiroki,
Ra Moonjin,
Samejima Keijiro,
Taguchi Ryo,
Kohda Kohfuku
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.3451
Subject(s) - chemistry , serine , derivatization , threonine , mass spectrometry , peptide , phosphorylation , matrix assisted laser desorption/ionization , chromatography , biochemistry , desorption , organic chemistry , adsorption
We describe a useful method for the efficient ionization and relative quantification of peptides containing serine/threonine phosphorylation sites. This method is based on β ‐elimination of the phosphate group from serine/threonine phosphorylation sites under alkaline conditions, followed by Michael addition reaction with N ‐(2‐mercaptoethyl)‐6‐methylnicotinamide (MEMN). As a result of the derivatization reaction, the negatively charged phosphate group is substituted with the nicotinoyl moiety to improve the ionization efficiency of the derivatized peptide. The combination of d 3 ‐labeled MEMN (d 3 ‐MEMN) and MEMN (d 0 ‐MEMN) generates a 3 Da mass difference between d 3 ‐MEMN‐labeled and d 0 ‐MEMN‐labeled peptides, which is a useful signature for the identification of peptides containing serine/threonine phosphorylation sites in the matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrum. Moreover, the mass difference is useful for the quantitative analysis of serine/threonine phosphorylation in proteins. In this paper, we describe the synthesis of d 0 /d 3 ‐labeled MEMN and an application of our approach to model peptides and proteins. Copyright © 2008 John Wiley & Sons, Ltd.