title: Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression changes.
abstract: The interpretation of quantitative phosphoproteomics studies is complicated because each differential phosphorylation event integrates both changes in protein expression and phosphorylation. Here we investigated this phenomenon by performing parallel comparisons of protein expression and phosphorylation in S. cerevisiae. In each of two experiments comparing yeast mutants bearing deletions in FUS3 or STE7 with their wild-type counterparts, we quantified over 4100 proteins, including all members of the yeast mating pathway. We also identified 12,499 unique phosphorylation sites in this work. We demonstrate the critical importance of controlling the protein-level false-discovery rate and provide a novel method to assess the accuracy of protein false-discovery rate estimates. For the first time, 96% of nonredundant phosphopeptide ratios could be calibrated by protein levels, allowing truly differential phosphorylation to be distinguished from altered protein expression. This revealed a starkly different view, with 25% of seemingly differential phosphopeptides now attributed to changes in protein expression. Combined protein expression and phosphorylation surveys uncovered both independent and concerted changes in protein expression and phosphorylation, while highlighting the partially redundant role of a second MAPK (Kss1) in the mating pathway.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Ronghu
authors.initials: R
authors.lastname: Wu
authors.forename: Noah
authors.initials: N
authors.lastname: Dephoure
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Haas
authors.forename: Edward L
authors.initials: EL
authors.lastname: Huttlin
authors.forename: Bo
authors.initials: B
authors.lastname: Zhai
authors.forename: Mathew E
authors.initials: ME
authors.lastname: Sowa
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
mesh_terms: D000595:Amino Acid Sequence; D002138:Calibration; D015870:Gene Expression; D055786:Gene Knockout Techniques; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D012100:Reproduction, Asexual; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C075580:FUS3 protein, S cerevisiae; C541984:KSS1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C119844:STE7 protein, S cerevisiae