Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry

  • Hannah Johnson
  • Claire E. Eyers
  • Patrick A. Eyers
  • Robert J. Beynon
  • Simon J. GaskellEmail author


Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment.


Tryptic Peptide Absolute Quantification Tryptic Digestion Phosphatase Treatment Reference Peptide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  • Hannah Johnson
    • 1
  • Claire E. Eyers
    • 1
  • Patrick A. Eyers
    • 2
  • Robert J. Beynon
    • 3
  • Simon J. Gaskell
    • 1
    Email author
  1. 1.Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of ChemistryUniversity of ManchesterManchesterUnited Kingdom
  2. 2.Yorkshire Cancer Research Institute for Cancer StudiesUniversity of SheffieldSheffieldUnited Kingdom
  3. 3.Proteomics and Functional Genomics Group, Faculty of Veterinary ScienceUniversity of LiverpoolLiverpoolUnited Kingdom

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