Abstract
The functional role of protein phosphorylation is impacted by its fractional stoichiometry. Thus, a comprehensive strategy to study phosphorylation dynamics should include an assessment of site stoichiometry. Here we report an integrated method that relies on phosphatase treatment and stable-isotope labeling to determine absolute stoichiometries of protein phosphorylation on a large scale. This approach requires the measurement of only a single ratio relating phosphatase-treated and mock-treated samples. Using this strategy we determined stoichiometries for 5,033 phosphorylation sites in triplicate analyses from Saccharomyces cerevisiae growing through mid-log phase. We validated stoichiometries at ten sites that represented the full range of values obtained using synthetic phosphopeptides and found excellent agreement. Using bioinformatics, we characterized the biological properties associated with phosphorylation sites with vastly differing absolute stoichiometries.
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Acknowledgements
This work was supported in part by US National Institutes of Health grants (HG3456) to S.P.G. We thank all members of the Gygi lab for help, especially R.A. Everley for his help with instrumentation and L. Ting for critically reading the manuscript.
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S.P.G. and R.W. designed the research. R.W., W.H., N.D., E.L.H., B.Z., M.E.S. and S.P.G. participated in the data generation, analysis and interpretation. R.W. and S.P.G. wrote the manuscript and all authors edited it.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4, Supplementary Tables 1 and 5 (PDF 381 kb)
Supplementary Table 2
Peptides identified in experiment 1. (XLSX 14310 kb)
Supplementary Table 3
Peptides identified in experiment 2. (XLSX 14521 kb)
Supplementary Table 4
Peptides identified in experiment 3. (XLSX 15000 kb)
Supplementary Table 6
Site stoichiometries obtained in biological triplicate experiments. (XLSX 584 kb)
Supplementary Table 7
Site stoichiometries for events described as Cdk1substrates. (XLSX 21 kb)
Supplementary Table 8
Examples of phosphorylation sites with high stoichiometries. (XLSX 11 kb)
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Wu, R., Haas, W., Dephoure, N. et al. A large-scale method to measure absolute protein phosphorylation stoichiometries. Nat Methods 8, 677–683 (2011). https://doi.org/10.1038/nmeth.1636
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DOI: https://doi.org/10.1038/nmeth.1636
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