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Related Experiment Video

Updated: Jun 18, 2026

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
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Stoichiometric quantification of Akt phosphorylation using LC-MS/MS.

Abdelmadjid Atrih1, Dan Turnock, Grant Sellar

  • 1Translational Medicine Research Collaboration, Dundee DD1 9SY, United Kingdom.

Journal of Proteome Research
|November 12, 2009
PubMed
Summary

A new liquid chromatography-mass spectrometry (LC-MS) method accurately quantifies Akt phosphorylation, a key indicator of the phosphoinositide 3-kinase (PI3-K) pathway. This technique offers superior accuracy and dynamic range compared to traditional Western blotting for cancer and diabetes research.

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Published on: November 15, 2017

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Mass Spectrometry

Background:

  • The phosphoinositide 3-kinase (PI3-K) pathway regulates critical cellular processes like survival, proliferation, and apoptosis.
  • Dysregulation of the PI3-K pathway is implicated in diseases including cancer and diabetes.
  • Quantifying Akt phosphorylation is a standard method to assess PI3-K pathway activity, traditionally done semiquantitatively via Western blot.

Purpose of the Study:

  • To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for accurate Akt phosphorylation stoichiometry measurement.
  • To compare the performance of the LC-MS/MS method against traditional Western blotting.

Main Methods:

  • Developed an LC-MS/MS procedure involving immunoprecipitation, gel electrophoresis, in-gel digestion, and addition of isotopically labeled internal standards.
  • Utilized chymotrypsin and trypsin for peptide fragmentation to measure Thr308 and Ser473 phosphorylation, respectively.
  • Validated the method on human T-cells and glioblastoma cells (U-87 MG) under various treatment conditions (pervanadate, PI3-K inhibitor).

Main Results:

  • The LC-MS/MS method demonstrated high interday precision (3.8% for Thr308, 2.3% for Ser473).
  • Untreated T-cells showed Akt phosphorylation below 1%; pervanadate treatment increased Thr308 phosphorylation 18-fold, partially reversed by a PI3-K inhibitor.
  • LC-MS/MS showed comparable sensitivity to Western blotting but offered superior accuracy, wider dynamic range, and more in-depth information.

Conclusions:

  • The developed LC-MS/MS method provides accurate quantification of Akt phosphorylation stoichiometry.
  • This method surpasses Western blotting in accuracy, dynamic range, and information depth for assessing PI3-K pathway activity.
  • The method holds potential for identifying predictive markers of response to targeted therapies in diseases like cancer.