Resolving protein–protein interactions (PPIs) inside biomolecular condensates is crucial for elucidating their functions and regulation mechanisms. The transient nature of condensates and the multiple localizations of clients, however, have rendered it challenging to determine compartment-specific PPIs. Here we developed a condensation-enhanced, spatially directed, metabolic incorporation-assisted photocrosslinking strategy—termed DenseMAP—for spatiotemporally resolved dissection of the direct protein interactome within condensates. By leveraging our condensation-enhanced photocrosslinker and the spatially directed biotin tagging, DenseMAP enabled stress-granule-specific interactome mapping of the N⁶-methyladenosine readers YTHDF1 and YTHDF2, and uncovered the functional role of phosphorylation on the SARS-CoV-2 nucleocapsid protein in regulating virus replication. Further applying DenseMAP for direct interactome mapping of the subcompartmental scaffold protein NPM1 deciphered nucleolar granular component proteome, and unveiled the critical role of SUMOylation in controlling nucleolar proteome homeostasis. DenseMAP provides a platform technology for analysing functional PPI networks within subcellular and subcompartmental condensates under diverse physiological and/or pathological settings.

解析生物分子凝聚物中的蛋白质-蛋白质相互作用 （PPI） 对于阐明其功能和调节机制至关重要。然而，冷凝物的瞬态性质和客户的多重定位使得确定隔室特定的 PPI 变得具有挑战性。在这里，我们开发了一种缩合增强、空间定向、代谢掺入辅助光交联策略——称为 DenseMAP——用于时空分辨缩合物内直接蛋白质相互作用组的解剖。通过利用我们的缩合增强光交联剂和空间定向生物素标记，DenseMAP 实现了 N⁶-甲基腺苷读取器 YTHDF1 和 YTHDF2 的应激颗粒特异性相互作用组图谱，并揭示了磷酸化对 SARS-CoV-2 核衣壳蛋白在调节病毒复制中的功能作用。进一步应用 DenseMAP 对亚区室支架蛋白 NPM1 进行直接相互作用组作图，破译了核仁颗粒成分蛋白质组，并揭示了 SUMOylation 在控制核仁蛋白质组稳态中的关键作用。DenseMAP 提供了一种平台技术，用于分析不同生理和/或病理环境下亚细胞和亚房凝聚物中的功能性 PPI 网络。