A large-scale method to measure absolute protein phosphorylation stoichiometries

The underlying 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 developed an integrated method that relies on phosphatase treatment and stable isotope labeling to determine the absolute stoichiometries of protein phosphorylation on a large-scale. This approach requires the measurement of only a single ratio relating phosphatase- and mock-treated samples. We applied the strategy to determine stoichiometries for 5,033 phosphorylation sites in Saccharomyces cerevisiae. Stoichiometries were determined from biological triplicate experiments with good reproducibility. We validated ten sites stoichiometries representing the full range of values with an absolute quantitative approach, showing excellent agreement. Using bioinformatics, we characterized the biological properties associated with phosphorylation sites with vastly differing absolute stoichiometries.