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Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS

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Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS. / Atrih, Abdelmadjid; Turnock, Dan; Sellar, Grant; Thompson, Alastair; Feuerstein, Giora; Ferguson, Michael A. J.; Huang, Jeffrey T-J.

In: Journal of Proteome Research, Vol. 9, No. 2, 02.2010, p. 743-751.

Research output: Contribution to journalArticle

Harvard

Atrih, A, Turnock, D, Sellar, G, Thompson, A, Feuerstein, G, Ferguson, MAJ & Huang, JT-J 2010, 'Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS' Journal of Proteome Research, vol 9, no. 2, pp. 743-751.

APA

Atrih, A., Turnock, D., Sellar, G., Thompson, A., Feuerstein, G., Ferguson, M. A. J., & Huang, J. T-J. (2010). Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS. Journal of Proteome Research, 9(2), 743-751doi: 10.1021/pr900572h

Vancouver

Atrih A, Turnock D, Sellar G, Thompson A, Feuerstein G, Ferguson MAJ et al. Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS. Journal of Proteome Research. 2010 Feb;9(2):743-751.

Author

Atrih, Abdelmadjid; Turnock, Dan; Sellar, Grant; Thompson, Alastair; Feuerstein, Giora; Ferguson, Michael A. J.; Huang, Jeffrey T-J. / Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS.

In: Journal of Proteome Research, Vol. 9, No. 2, 02.2010, p. 743-751.

Research output: Contribution to journalArticle

Bibtex - Download

@article{7915b25035394f4aaadbc8f6e43026b1,
title = "Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS",
author = "Abdelmadjid Atrih and Dan Turnock and Grant Sellar and Alastair Thompson and Giora Feuerstein and Ferguson, {Michael A. J.} and Huang, {Jeffrey T-J.}",
year = "2010",
volume = "9",
number = "2",
pages = "743--751",
journal = "Journal of Proteome Research",
issn = "1535-3893",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS

A1 - Atrih,Abdelmadjid

A1 - Turnock,Dan

A1 - Sellar,Grant

A1 - Thompson,Alastair

A1 - Feuerstein,Giora

A1 - Ferguson,Michael A. J.

A1 - Huang,Jeffrey T-J.

AU - Atrih,Abdelmadjid

AU - Turnock,Dan

AU - Sellar,Grant

AU - Thompson,Alastair

AU - Feuerstein,Giora

AU - Ferguson,Michael A. J.

AU - Huang,Jeffrey T-J.

PY - 2010/2

Y1 - 2010/2

N2 - <p>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.</p>

AB - <p>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.</p>

KW - Akt

KW - PKB

KW - phosphorylation

KW - stoichiometry

KW - LC-MS/MS

KW - T cells

KW - U-87 MG cells

KW - MONITORING MASS-SPECTROMETRY

KW - AKT/PROTEIN-KINASE-B

KW - ABSOLUTE QUANTIFICATION

KW - PROTEIN-KINASE

KW - HUMAN CANCER

KW - PATHWAY

KW - ACTIVATION

KW - MTOR

KW - GROWTH

KW - METABOLISM

U2 - 10.1021/pr900572h

DO - 10.1021/pr900572h

M1 - Article

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 2

VL - 9

SP - 743

EP - 751

ER -

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