Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry.
Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry.
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DOI：
10.1038/s41596-021-00604-3
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发表时间：
2021-10
期刊：
影响因子：
14.8
通讯作者：
Kettenbach AN
中科院分区：
文献类型：
--
作者：
Brauer BL;Wiredu K;Mitchell S;Moorhead GB;Gerber SA;Kettenbach AN
磷酸蛋白磷酸酶（PPPs）在细胞和组织中执行超过90%的丝氨酸/苏氨酸去磷酸化。虽然PPP在细胞生物学和疾病（如癌症，心脏肥大和阿尔茨海默病）中的作用已经得到了很好的证实，但PPP的分子机制仍有待发现。在这里，我们描述了一种化学蛋白质组学策略，磷酸酶抑制剂珠和质谱（PIB-MS），使识别和定量的PPP及其翻译后修饰在短短12小时。使用固定在珠上的特异性但非选择性的PPP抑制剂，PIB-MS使得能够从细胞和组织中有效地亲和捕获、鉴定和定量内源性PPP和相关蛋白（“PPPome”）。PIB-MS捕获功能性的内源性PPP亚基相互作用，并允许发现新的结合伴侣。它进行PPP富集，而无需外源表达标记蛋白或特异性抗体。由于PPP是进化过程中最保守的蛋白质之一，因此PIB-MS可用于任何细胞系，组织或生物体。该方案描述了一种蛋白质组学方法，用于有效的亲和捕获、鉴定和定量来自细胞和组织的内源性磷蛋白磷酸酶和相关蛋白。细胞和组织内源性磷蛋白磷酸酶和相关蛋白的亲和捕获和蛋白质组学分析的新方案。内源性磷蛋白磷酸酶的分析使用该方案的关键参考文献里昂，S.等人MCP 17，2448-2461（2018）：https://doi.org/10.1074/mcp.RA118.000822等人Science Signaling 13，eaba 7823（2020）：https://doi.org/10.1126/scisignal.aba7823
Phosphoprotein Phosphatases (PPPs) execute over 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 post-translational modifications in as little as 12 hours. Using a specific but non-selective 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 allows for discovering 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. This protocol describes a proteomic approach for efficient affinity-capture, identification, and quantification of endogenous phosphoprotein phosphatases and associated proteins from cells and tissues. New protocol for affinity capture and proteomic analysis of endogenous phosphoprotein phosphatases and associated proteins from cells and tissues. Profiling endogenous phosphoprotein phosphatases Key references using this protocol Lyons, S. P. et al. MCP 17, 2448–2461 (2018): https://doi.org/10.1074/mcp.RA118.000822 Nasa, I. et al. Science Signaling 13, eaba7823 (2020): https://doi.org/10.1126/scisignal.aba7823
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