Hi-APEX enables hydrogen peroxide-independent proximity labeling through APEX2-catalyzed tetrazine-phenol radical formation, eliminating oxidative artifacts to reveal authentic molecular compositions of redox-sensitive processes like stress granules and ferroptosis, and achieving the first genuine in vivo proximity labeling in living animals including tumor xenografts and hippocampal neurons.

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.