Journal Article Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.

Tsai, Chia-Feng  ,  Wang, Yi-Ting  ,  Yen, Hsin-Yung  ,  Tsou, Chih-Chiang  ,  Ku, Wei-Chi  ,  Lin, Pei-Yi  ,  Chen, Hsuan-Yu  ,  Nesvizhskii, Alexey I  ,  Ishihama, Yasushi  ,  Chen, Yu-Ju

62015-03-27 , Nature Publishing Group
Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targeting assays in lung cancer cells demonstrate the advantage of kinase-targeted complexity reduction, resulting in deeper phosphoproteome quantification. We measure the phosphorylation stoichiometry of >1,000 phosphorylation sites including 366 low-abundance tyrosine phosphorylation sites, with high reproducibility and using small sample sizes. Comparing drug-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and suggest potential drug targets within the kinase-substrate network associated with acquired drug resistance.

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