Details

Autor(en) / Beteiligte

• Sharma, Kirti
• D’Souza, Rochelle C.J.
• Tyanova, Stefka
• Schaab, Christoph
• Wiśniewski, Jacek R.
• Cox, Jürgen
• Mann, Matthias

Titel

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

Ist Teil von

• Cell reports (Cambridge), 2014-09, Vol.8 (5), p.1583-1594

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2014

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 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 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. [Display omitted] •Deepest phosphoproteome with more than 50,000 distinct phosphopeptides•Stringent computational pipeline for high-quality phosphoproteomics analysis•Large-scale phosphosite occupancies extracted from label-free data•At least 75% of the proteome phosphorylated following an 80/20 rule Here, Sharma et al. use mass spectrometry to map the phosphoproteome to a depth of about 50,000 distinct phosphopeptides. They show that at least 75% of the proteome is phosphorylated following an 80/20 rule and that specific signaling states are characterized by high fractional site occupancies. They uncover fundamental differences between Ser/Thr and Tyr phosphorylation. Notably, they find that pTyr is much more tightly controlled and tends to occur on higher-abundance proteins and that the pTyr proteome appears to be “finite.”