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A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

Abstract

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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Fig. 1: Overview of hydrogen peroxide detection with Peroxymycin-1 in cells and mice.
Fig. 2: Synthetic and mechanistic scheme of Peroxymycin-1.
Fig. 3: In vitro analysis of Peroxymycin-1 reactivity and selectivity.
Fig. 4: Confocal microscopy images of endogenous H2O2 detection by using Peroxymycin-1.
Fig. 5: Profiling H2O2 levels with Peroxymycin-1 in liver tissue of mice fed a normal diet and a high-fat diet.

Data availability

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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Acknowledgements

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.

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K.H., J.O., C.Y.-S.C., M.S.M. and C.J.C. wrote the manuscript. K.H. and J.O. developed the new synthesis. C.Y.-S.C. performed the imaging experiments. M.S.M. reproduced the synthesis and provided full characterization of the compounds. M.S.M. performed imaging experiments required for revisions.

Corresponding author

Correspondence to Christopher J. Chang.

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A patent application has been filed for the Peroxymycin-1 probe. The patent application number is PCT/US2019/023242.

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Nature Protocols thanks Deju Ye and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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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

Supplementary information

Supplementary Information

Supplementary Text and Supplementary Figs. 1–8.

Source data

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 (2022). https://doi.org/10.1038/s41596-022-00694-7

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