Open AccessAffinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry
Author(s) -
Brooke L. Brauer,
Kwame Wiredu,
Sierra Mitchell,
Greg B. G. Moorhead,
Scott A. Gerber,
Arminja N. Kettenbach
Publication year - 2021
Publication title -
nature protocols
Language(s) - English
Resource type - Journals
eISSN - 1754-2189
pISSN - 1750-2799
DOI - 10.1038/s41596-021-00604-3
Subject(s) - phosphoprotein , interactome , endogeny , phosphatase , dephosphorylation , proteomics , phosphorylation , chemistry , biochemistry , phosphoproteomics , biology , microbiology and biotechnology , protein phosphorylation , computational biology , protein kinase a , gene
Phosphoprotein phosphatases (PPPs) execute >90% of serine/threonine dephosphorylation in cells and tissues. While the role of PPPs in cell biology and diseases such as cancer, cardiac hypertrophy and Alzheimer's disease is well established, the molecular mechanisms governing and governed by PPPs still await discovery. Here we describe a chemical proteomic strategy, phosphatase inhibitor beads and mass spectrometry (PIB-MS), that enables the identification and quantification of PPPs and their posttranslational modifications in as little as 12 h. Using a specific but nonselective PPP inhibitor immobilized on beads, PIB-MS enables the efficient affinity-capture, identification and quantification of endogenous PPPs and associated proteins ('PPPome') from cells and tissues. PIB-MS captures functional, endogenous PPP subunit interactions and enables discovery of new binding partners. It performs PPP enrichment without exogenous expression of tagged proteins or specific antibodies. Because PPPs are among the most conserved proteins across evolution, PIB-MS can be employed in any cell line, tissue or organism.