Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling

Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling

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Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells.Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown.Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line.We detected more than three-quarters of cellular proteins as phosphoproteins and determined Outdoor Dining Arm Chair (Set of 2) very high stoichiometries in mitosis or growth factor signaling by label-free quantitation.The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons.

Tyrosine phosphorylation is maintained at especially low stoichiometric levels Easy Straight Crop Jean in the absence of specific signaling events.Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases.Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.