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

Chen, B.; Guo, H.; Yan, Z.; Lu, W.; Li, C.; Xu, S.; Zhang, Y.; Sun, S.; Sun, X.; Zhao, S.; Shangguan, Q.; Chen, Y.; Lu, L.; Wu, Z.; Qin, W.

doi:10.64898/2026.03.13.711744

bioRxiv · 10.64898/2026.03.13.711744

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

Chen, B.·Guo, H.·Yan, Z.·Lu, W.·Li, C.·Xu, S.·Zhang, Y.·Sun, S.·Sun, X.·Zhao, S.·Shangguan, Q.·Chen, Y.·Lu, L.·Wu, Z.·Qin, W.

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Abstract

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 probes 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 m{superscript 1}A 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.

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Chen, B., Guo, H., Yan, Z., Lu, W., Li, C., Xu, S., Zhang, Y., Sun, S., Sun, X., Zhao, S., Shangguan, Q., Chen, Y., Lu, L., Wu, Z., Qin, W.. 2026-03-15. In vivo-compatible spatial multi-omics via hydrogen peroxide-independent APEX2 labeling. https://doi.org/10.64898/2026.03.13.711744

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