Phosphorylation stoichiometry measurement on a large scale by motif-targeting quantitative proteomics

摘要

To our knowledge, this is the first large-scale approach to assess the phosphorylation stoichiometry of a single-state human proteome. The proof-of-concept experiments on CK2-, MAPK- and EGFR-targeted assays show the advantages of kinase specificity-based complexity reduction of phosphopeptides, which further demonstrates efficient enrichment of low abundant phosphorylation sites. Such kinase-targeted assays also show high sensitivity for determining the stoichiometry of hundreds of tyrosine phosphorylation sites in 50 μg of cell lysate. In a single cellular state, our results revealed that distinct phosphorylation stoichiometry distributions of different substrates and motifs can be distinguished. Through quantitative measurement of the drug-resistant and sensitive lung cancer cells, this motif-targeting approach suggested potential drug-targeting proteins in the EGFR- and CK2-centered kinase-substrate network. This approach can provide system-wide maps of protein phosphorylation stoichiometry for either single or multiple cellular states under physiological or pathological regulation.