title: Stoichiometric quantification of Akt phosphorylation using LC-MS/MS.
abstract: 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.
authors.affiliation: Translational Medicine Research Collaboration, Dundee DD1 9SY, United Kingdom.
authors.forename: Abdelmadjid
authors.initials: A
authors.lastname: Atrih
authors.forename: Dan
authors.initials: D
authors.lastname: Turnock
authors.forename: Grant
authors.initials: G
authors.lastname: Sellar
authors.forename: Alastair
authors.initials: A
authors.lastname: Thompson
authors.forename: Giora
authors.initials: G
authors.lastname: Feuerstein
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
authors.forename: Jeffrey T-J
authors.initials: JT
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010455:Peptides; D051057:Proto-Oncogene Proteins c-akt