Stefka Tyanova

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.

Mass spectrometry-based proteomics is a continuously growing field marked by technological and methodological improvements. Cancer proteomics is aimed at pursuing goals such as accurate diagnosis, patient stratification, and biomarker discovery, relying on the richness of information of quantitative proteome profiles. Translating these high-dimensional data into biological findings of clinical importance necessitates the use of robust and powerful computational tools and methods. In this chapter, we provide a detailed description of standard analysis steps for a clinical proteomics dataset performed in Perseus, a software for functional analysis of large-scale quantitative omics data.