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

• Published in: Nature communications 2015-03, Vol.6 (1), p.6622-6622, Article 6622
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: 631/45/475; 631/553; 631/80/458/1733; 692/699/67/1612; 96/95; Algorithms; Amino Acid Motifs; Blotting, Western; Cancer; Cell Line, Tumor; Chromatography, Liquid; Dephosphorylation; Drug resistance; Drug Resistance, Neoplasm; Epidermal growth factor receptors; Humanities and Social Sciences; Humans; Kinases; Lung cancer; Lung Neoplasms - metabolism; MAP kinase; mRNA; multidisciplinary; Phosphoproteins - metabolism; Phosphorylation; Post-translation; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proteins; Proteomics; Quantitation; Reproducibility; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA, Messenger - metabolism; Science; Science (multidisciplinary); Signal Transduction; Stoichiometry; Substrates; Tandem Mass Spectrometry; Typing; Tyrosine; Workflow
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms7622

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. Measuring phosphorylation stoichiometry on a proteomic scale remains a challenge. Tsai et al . develop a technique to measure the basal level of phosphorylation stoichiometry in a single human phosphoproteome and identify molecular changes associated with gefitinib resistance in lung cancer cells.