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.

磷蛋白磷酸酶 (PPP) 在细胞和组织中执行 >90% 的丝氨酸/苏氨酸去磷酸化。虽然 PPP 在细胞生物学和癌症、心脏肥大和阿尔茨海默氏病等疾病中的作用已经确定，但 PPP 控制和控制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法 (PIB-MS)，能够在短短 12 小时内识别和量化 PPP 及其翻译后修饰。PIB-MS 使用固定在珠子上的特异性但非选择性 PPP 抑制剂，能够有效地亲和捕获、鉴定和定量来自细胞和组织的内源性 PPP 和相关蛋白 ('PPPome')。PIB-MS 捕获功能，内源性 PPP 亚基相互作用并能够发现新的结合伙伴。它无需外源表达标记蛋白或特异性抗体即可进行 PPP 富集。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。