Abstract
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.
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Acknowledgements
A.N.K. and S.A.G. acknowledge support from NCI R33 CA225458. S.A.G. received support from NCI P30 CA0231008. K.W. was supported by a Burroughs-Wellcome Big Data in the Life Sciences Fellowship. We thank K. Smolen for assistance with video editing.
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B.L.B., S.M., G.B.M. and A.N.K. developed and wrote the protocol. B.L.B. drew the illustrations. K.W. and S.A.G. contributed to the design of the data analysis workflow. S.A.G. contributed to the editing of the manuscript.
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Peer review information Nature Protocols thanks Albert J. R. Heck, Martin R. Larsen and the other, anonymous reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Lyons, S. P. et al. Mol. Cell Proteomics 17, 2448–2461 (2018): https://doi.org/10.1074/mcp.RA118.000822
Nasa, I. et al. Sci. Signal. 13, eaba7823 (2020): https://doi.org/10.1126/scisignal.aba7823
Supplementary information
Supplementary Table 1
Annotated PPP subunits and interactors. Uniprot ID and entry name are given for each protein, alongside its gene name and description. PPP subunit indicates the PPP the proteins belong to (PP1-7), whether it is a catalytic subunit (C), a regulatory subunit (R), an inhibitory protein (i), or an activating protein (a).
Supplementary Video 1
A step-by-step demonstration of making a StageTip and using it to desalt a TMT label check sample.
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Brauer, B.L., Wiredu, K., Mitchell, S. et al. Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry. Nat Protoc 16, 4919–4943 (2021). https://doi.org/10.1038/s41596-021-00604-3
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DOI: https://doi.org/10.1038/s41596-021-00604-3
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