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

TLDR

Regulatory protein phosphorylation governs normal and pathological signaling in eukaryotic cells, yet its extent, localization, and site‑specific stoichiometry remain largely unknown despite advances in mass‑spectrometry‑based proteomics. The study aims to develop a stringent experimental and computational workflow capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. The authors employ this workflow, combining rigorous experimental procedures with computational analysis, to identify over 50,000 unique phosphopeptides in the cell line. The resulting phosphoproteome shows that more than three‑quarters of cellular proteins are phosphorylated, with high stoichiometry in mitosis or growth‑factor signaling; phospho‑tyrosine sites are scarce, low‑stoichiometric outside specific signaling, enriched on high‑abundance proteins, and correlate with tyrosine‑kinase substrate KM values, indicating that P‑Tyr is a functionally distinct PTM.

Abstract

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.