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

This article describes the identification of the autophosphorylation sites of the G protein-sensitive class I phosphoinositide 3-kinase isoforms β and γ 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 32P-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 p110β and p110γ subunits could be unambiguously assigned to the C-terminal Ser 1070 and Ser 1101, respectively. Copyright © 2003 John Wiley & Sons, Ltd.