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Bioscience Reports (2010) Immediate Publication, doi:10.1042/BSR20090167
Electron Capture Dissociation Mass Spectrometric Analysis of Lysine-Phosphorylated Peptides
Karolina Kowalewska, Piotr Stefanowicz, Tomasz Ruman, Tomasz Frączyk, Wojciech K Rode and Zbigniew Szewczuk
Faculty of Chemistry, University of Wroclaw, WROCLAW PL-50-383, Poland. szewczuk@wchuwr.pl
Phosphorylation of proteins is an essential signaling mechanism in eukaryotic and prokaryotic cells. Although N-phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In this study, two mass spectrometry fragmentation methods, electron-capture dissociation (ECD) and collision-induced dissociation (CID), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE, and PLSRTLSVAAKK. The peptides were phosphorylated by the reaction with monopotassium phosphoramidate. The results were confirmed by the 1H NMR and 31P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to phosphoramidate moiety. Moreover, the results indicate that the ECD mass spectrometry may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested.
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