In vivo -compatible spatial multi-omics via hydrogen peroxide-independent APEX2 labeling

Proximity labeling (PL) technologies like APEX2 have transformed spatial multi-omics in live cells, but their long-standing dependence on hydrogen peroxide (H₂O₂) disrupts redox signaling and prevents use in live animals. Here we introduce H₂O₂-independent APEX2 (Hi-APEX), which uses a clickable tetrazine-phenol probe, requiring no enzyme engineering. We show that APEX2 directly catalyzes TP radical formation without H₂O₂ via a mechanism requiring the probe’s tetrazine group and a key histidine residue. We benchmarked Hi-APEX-based spatial multi-omics by mapping the mitochondrial matrix and dynamic secretomes. Hi-APEX significantly outperforms traditional APEX in capturing redox-sensitive processes such as stress response and ferroptosis, enabling discovering authentic stress granule components and protein interaction networks for mitochondria-localized GPx4. One mGPx4 interactor TRMT61B—known to regulate mitochondrial m¹A modifications—promotes ferroptosis. Crucially, Hi-APEX achieves full in vivo compatibility, enabling direct PL in tumor xenografts and hippocampal neurons, thereby expanding PL-based spatial multi-omics from cellular systems to living organisms.