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

Here we introduce H₂O₂-independent APEX2 (Hi-APEX), which uses a clickable tetrazine-phenol (TP) probe, requiring no enzyme engineering. We show that APEX2 directly catalyzes TP labeling without H₂O₂ via a mechanism requiring the probe's tetrazine group and oxygen.

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 (mGPX4). One mGPX4 interactor TRMT61B—known to regulate mitochondrial m¹A modifications—promotes ferroptosis.