Premium
Dynamic identification of phosphopeptides using immobilized metal ion affinity chromatography enrichment, subsequent partial β ‐elimination/chemical tagging and matrix‐assisted laser desorption/ionization mass spectrometric analysis
Author(s) -
Ahn Yeong Hee,
Park Eun Jung,
Cho Kun,
Kim Jin Young,
Ha Sang Hoon,
Ryu Sung Ho,
Yoo Jong Shin
Publication year - 2004
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.1651
Subject(s) - phosphopeptide , chemistry , nitrilotriacetic acid , chromatography , mass spectrometry , peptide , combinatorial chemistry , organic chemistry , biochemistry , chelation
The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified β ‐elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time‐dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol‐tagged derivatives simultaneously by their mass difference (–20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data. Copyright © 2004 John Wiley & Sons, Ltd.