title: A large-scale method to measure absolute protein phosphorylation stoichiometries.
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.
authors: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors: Ronghu
authors: R
authors: Wu
authors: Wilhelm
authors: W
authors: Haas
authors: Noah
authors: N
authors: Dephoure
authors: Edward L
authors: EL
authors: Huttlin
authors: Bo
authors: B
authors: Zhai
authors: Mathew E
authors: ME
authors: Sowa
authors: Steven P
authors: SP
authors: Gygi
mesh_terms: D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology*; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D020543:Proteome; D010744:Phosphoric Monoester Hydrolases