Premium
Stoichiometric Quantification of Akt Phosphorylation using LC‐MS/MS
Author(s) -
Atrih Abdelmadjid,
Turnock Dan,
Sellar Grant,
Thompson Alastair,
Ferguson Michael,
Huang Jeffrey
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.902.1
Subject(s) - phosphorylation , chemistry , protein kinase b , trypsin , kinase , peptide , chromatography , mass spectrometry , blot , protein phosphorylation , biochemistry , microbiology and biotechnology , enzyme , biology , protein kinase a , gene
Quantification of phospho‐Akt is a standard way of assessing the activity of the Ptdlns‐3‐kinase signalling pathway. Here we report an LC‐MS method to measure the stoichiometry of Akt phosphorylation in biological samples with good precision. The procedure includes immunoprecipitation, gel electrophoresis, in‐gel digestion, addition of stable isotope labelled internal standards and LC‐MS/MS. Two proteolytic enzymes, chymotrypsin and trypsin, were used to generate suitable peptide fragments for measuring Thr308 and Ser473 phosphorylation, respectively. The inter‐day imprecision was estimated to be 3.8% and 2.3% for Thr308 and Ser473, respectively. This method has been tested on human T‐cells grown in presence and absence of pervanadate with or without a PI3‐K inhibitor. The results suggest that the levels of Akt phosphorylation in untreated cells were below 1% for both phosphorylation sites. Pervanadate treatment provoked a 39‐ and 18‐fold increase of phosphorylation at Thr308 and Ser473, respectively. A comparison between LC‐MS/MS and Western blotting suggests that the LC‐MS based method provides a more accurate phosphorylation stoichiometry and more in‐depth information.