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

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Abstract
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.