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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.

SUBMITTER: Tsai CF 

PROVIDER: S-EPMC4389224 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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

Tsai Chia-Feng CF   Wang Yi-Ting YT   Yen Hsin-Yung HY   Tsou Chih-Chiang CC   Ku Wei-Chi WC   Lin Pei-Yi PY   Chen Hsuan-Yu HY   Nesvizhskii Alexey I AI   Ishihama Yasushi Y   Chen Yu-Ju YJ  

Nature communications 20150327


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-targ  ...[more]

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