phosphoproteomic-strategy-for-profiling-osmotic-stress-signaling

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in <em>Arabidopsis</em>

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Kyoto University

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.

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

Direct measurement of site-specific phosphorylation stoichiometry can unambiguously distinguish whether the degree of phosphorylation is regulated by upstream kinase/phosphatase activity or by transcriptional regulation to alter protein expression level. Here, we describe a motif-targeting quantitative proteomic approach that integrates dephosphorylation, isotope tag labeling, and enzymatic kinase reaction for large-scale phosphorylation stoichiometry measurement of the human proteome.

Article	Year
Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics. Nature communications\| PubMed ID: 25814448	2015
Absolute Phosphorylation Stoichiometry Analysis by Motif-Targeting Quantitative Mass Spectrometry. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28730488	2017

Chia-Feng Tsai

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