title: Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.
abstract: 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.
authors: Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.|Institute of Chemistry, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.
authors: Chia-Feng
authors: CF
authors: Tsai
authors: Institute of Chemistry, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.|Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.|Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan.
authors: Yi-Ting
authors: YT
authors: Wang
authors: Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan.|Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.
authors: Hsin-Yung
authors: HY
authors: Yen
authors: Department of Computational Medicine and Bioinformatics and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors: Chih-Chiang
authors: CC
authors: Tsou
authors: School of Medicine, Fu Jen Catholic University, New Taipei City 24257, Taiwan.
authors: Wei-Chi
authors: WC
authors: Ku
authors: Institute of Chemistry, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.
authors: Pei-Yi
authors: PY
authors: Lin
authors: Institute of Statistical Science, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.
authors: Hsuan-Yu
authors: HY
authors: Chen
authors: Department of Computational Medicine and Bioinformatics and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors: Alexey I
authors: AI
authors: Nesvizhskii
authors: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors: Yasushi
authors: Y
authors: Ishihama
authors: Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.|Institute of Chemistry, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.|Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, 128 Academia Road, Section 2, Taipei 11529, Taiwan.
authors: Yu-Ju
authors: YJ
authors: Chen
mesh_terms: D000465:Algorithms; D020816:Amino Acid Motifs; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D019008:Drug Resistance, Neoplasm; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: D010750:Phosphoproteins; D012333:RNA, Messenger