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 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 (32)P-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.
Czupalla
Cornelia
C
Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, Universitätsstr 1, Gebäude 22.03, 40225 Düsseldorf, Germany.
Nürnberg
Bernd
B
Krause
Eberhard
E
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D007527:Isoenzymes / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000145:classification* / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D010768:Phosphoserine; D019869:Phosphatidylinositol 3-Kinases; D014357:Trypsin