Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry |
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Authors: | Hannah Johnson Claire E Eyers Patrick A Eyers Robert J Beynon Simon J Gaskell |
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Institution: | 1. Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester, United Kingdom 2. Yorkshire Cancer Research Institute for Cancer Studies, University of Sheffield, Sheffield, United Kingdom 3. Proteomics and Functional Genomics Group, Faculty of Veterinary Science, University of Liverpool, Liverpool, United Kingdom
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Abstract: | 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. |
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