Analysis of class I phosphoinositide 3-kinase autophosphorylation sites by mass spectrometry

被引:13
作者
Czupalla, C
Nürnberg, B
Krause, E
机构
[1] Forschungsinst Mol Pharmakol, D-13125 Berlin, Germany
[2] Klinikum Heinrich Heine Univ, Inst Physiol Chem 2, D-40225 Dusseldorf, Germany
[3] Free Univ Berlin, Inst Pharmakol, D-14195 Berlin, Germany
关键词
D O I
10.1002/rcm.967
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
This article describes the identification of the autophosphorylation sites of the G protein-sensitive class I phosphoinositide 3-kinase isoforms beta and gamma by mass spectrometry. Since discrimination and suppression effects prevented the immediate detection and sequencing of phosphopeptides in complex mixtures, a strategy was applied that involved P-32-radiolabeling of the phosphoproteins, cleavage of the phosphoproteins with several proteases and/or cyanogen bromide, separation of the resulting peptide mixtures by micro-reversed-phase liquid chromatography, and mass spectrometric analysis of fractions containing phosphopeptides. As a result the primary autophosphorylation sites of phosphoinositide 3-kinase p110beta and p110gamma subunits could be unambiguously assigned to the C-terminal Ser 1070 and Ser 1101, respectively. Copyright (C) 2003 John Wiley Sons, Ltd.
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收藏
页码:690 / 696
页数:7
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