Hydrogen peroxide (H2O2) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H2O2 biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity–based sensing probe that responds to H2O2 via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H2O2 levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H2O2 levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3–4 d.
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Data are available through figshare: Fig. 2c, https://doi.org/10.6084/m9.figshare.16639975; Fig. 3a, https://doi.org/10.6084/m9.figshare.16639570; Fig. 4, https://doi.org/10.6084/m9.figshare.16640434; Fig. 5, https://doi.org/10.6084/m9.figshare.16639537; Supplementary Fig. 1, https://doi.org/10.6084/m9.figshare.18480879; Supplementary Fig. 2, https://doi.org/10.6084/m9.figshare.18480885; Supplementary Fig. 3, https://doi.org/10.6084/m9.figshare.18480888; Supplementary Fig. 4, https://doi.org/10.6084/m9.figshare.18480891; Supplementary Fig. 5, https://doi.org/10.6084/m9.figshare.18480894; Supplementary Fig. 6, https://doi.org/10.6084/m9.figshare.18480897; Supplementary Fig. 7, https://doi.org/10.6084/m9.figshare.18480900; Supplementary Fig. 8, https://doi.org/10.6084/m9.figshare.18480903. Source data are provided with this paper.
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We thank the NIH (R01 GM 79465, R01 GM 139245 and R01 ES 28096 to C.J.C.) for research support. K.H. thanks the College of Chemistry for a summer undergraduate research fellowship. J.O. thanks the Japan Society for the Promotion of Science for a postdoctoral fellowship. C.Y.-S.C. thanks the Croucher Foundation for a postdoctoral fellowship. M.S.M. thanks the UC President’s Postdoctoral Fellowship Program, Chinook-Berkeley Postdoctoral Fellowship Program and an NIH MOSAIC K99/R00 (1K99GM143573-01) award for funding. C.J.C. is a CIFAR Fellow. We thank Alison Killilea and Carissa Tasto (UC Berkeley Tissue Culture Facility) for expert technical assistance.
A patent application has been filed for the Peroxymycin-1 probe. The patent application number is PCT/US2019/023242.
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Key references using this protocol
Chung, C. et al. J. Am. Chem. Soc. 140, 6109–6121 (2018): https://doi.org/10.1021/jacs.8b02279
Spangler, B. et al. Nat. Chem. Biol. 12, 680–685 (2016): https://doi.org/10.1038/nchembio.2116
Source Data Fig. 3
Unprocessed HPLC data
Source Data Fig. 4
Fluorescence intensity data and statistical analysis
Source Data Fig. 5
Fluorescence intensity data and statistical analysis
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Hoshi, K., Messina, M.S., Ohata, J. et al. A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues. Nat Protoc 17, 1691–1710 (2022). https://doi.org/10.1038/s41596-022-00694-7
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