Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS

Abdelmadjid Atrih, Dan Turnock, Grant Sellar, Alastair Thompson, Giora Feuerstein, Michael A. J. Ferguson (Lead / Corresponding author), Jeffrey T-J. Huang (Lead / Corresponding author)

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    20 Citations (Scopus)


    The Ptdlns-3-kinase (PI3-K) signaling pathway plays a vital role in cell survival, proliferation, apoptosis and differentiation in normal cells, as well as in diseases such as cancer and diabetes. Quantification of phospho-Akt is a standard way of assessing the activity of the PI3-K signaling pathway in both cells and tumors. This measurement is traditionally performed semiquantitatively using immunoassays such as Western blot. Here we report an LC-MS method to accurately measure the stoichiometry of Akt phosphorylation in biological samples. The procedure includes immunoprecipitation, gel electrophoresis, in-gel digestion, addition of isotopicaly labeled 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 interday 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 and with or without a PI3-K inhibitor and on human glioblastoma cells (U-87 MG) grown in presence and absence of wortmannin (PI3-K inhibitor). The results of T cells suggest that the levels of Akt phosphorylation in untreated cells were below 1% for both phosphorylation sites. Pervanadate treatment provoked an 18-fold increase in phosphorylation of Thr308 and the PI3-K inhibitor partially reversed the increase. A comparison between LC-MS/MS and Western blotting suggests that the LC-MS based method is of comparable sensitivity and provides a more accurate phosphorylation stoichiometry, a wider dynamic range and more in-depth information. The application of the new method and its utility to providing predictive markers of response to targeted therapies is discussed.

    Original languageEnglish
    Pages (from-to)743-751
    Number of pages9
    JournalJournal of Proteome Research
    Issue number2
    Publication statusPublished - Feb 2010


    • Akt
    • PKB
    • phosphorylation
    • stoichiometry
    • LC-MS/MS
    • T cells
    • U-87 MG cells
    • MTOR
    • GROWTH


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