Organelle-Specific Interactome Mapping via Enzyme-Responsive Cross-Linking and Proximity Labeling

Chemical cross-linking mass spectrometry (CXMS) is a powerful technique for deciphering protein structures and interaction networks. Key frontiers in advancing CXMS for comprehensive biological insights include expanding the reactivity of cross-linkers beyond conventional amino acids as well as achieving organelle-specific cross-linking. Here, we present an enzyme-responsive cross-linking strategy that integrates lysine- and tyrosine-targeting cross-linking with proximity labeling. By leveraging an organelle-localized APEX2 peroxidase, this approach simultaneously activates phenol moieties for rapid in situ protein cross-linking and promotes protein biotinylation for subsequent enrichment and mass spectrometry analysis. We systematically validated the efficacy of this method using in vitro model proteins and live mammalian cells. Notably, the strategy enables nucleus-specific cross-linking with subcellular precision, establishing a robust platform for spatially resolved interactome mapping. Collectively, this study provides new opportunities to investigate dynamic protein interactions and subcellular organization with enhanced spatiotemporal resolution.