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Proteome-wide profiling of S-nitrosylated proteins using the SNOTRAP probe and mass spectrometry-based detection

Nature Protocols (2025)Cite this article

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Abstract

Protein S-nitrosylation (SNO) is a ubiquitous post-translational modification, which regulates a broad range of functional parameters, including protein stability; enzymatic, transcriptional and ion channel activity; and cellular signal transduction. Aberrant protein SNO is associated with diverse pathophysiology, from cardiovascular, metabolic and respiratory disorders to neurodegeneration and cancer. Drugs that enhance or inhibit specific SNO reactions are being developed as potential disease-modifying therapeutics. However, owing to a lack of suitable approaches to monitor SNO proteins, which often exist at low abundance with ephemeral expression, a systematic understanding of their roles in disease remains elusive. Here we report a robust and proteome-wide approach for the exploration of the S-nitrosoproteome in human and mouse tissues, using the brain as an example, with a probe named SNOTRAP (a triphenylphosphine thioester linked to a biotin molecule through a polyethylene glycol spacer group) in conjunction with mass spectrometry (MS)-based detection. In this Protocol, we detail tissue sample preparation, synthesis of SNOTRAP under an argon atmosphere and subsequent MS-based identification and analysis of SNO proteins. In situ labeling of SNO proteins is achieved by the SNOTRAP probe, concomitantly yielding a disulfide–iminophosphorane as a labeling tag. The chemically tagged proteins can be digested, followed by streptavidin capture, release by triscarboxyethylphosphine and relabeling of the liberated free Cys with N-ethylmaleimide. This approach selectively enriches SNO-containing peptides at specific sites for label-free quantification by Orbitrap MS. It requires about 5 d for synthesis of the SNOTRAP probe, 2–2.5 d for sample preparation and about 5 d for nano-liquid chromatography–tandem MS measurement and analysis.

Key points

  • This protocol for the exploration of the S-nitrosoproteome in human and mouse tissues identifies and quantifies S-nitrosylation (SNO)-containing peptides using the SNOTRAP probe, which selectively and specifically reacts with the SNO group, followed by nano-liquid chromatography–tandem mass spectrometry analysis.

  • The approach permits efficient and high-throughput proteome-wide profiling of SNO proteins in complex mixtures of biological material.

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Fig. 1: Integrative SNOTRAP proteome workflow to detect SNO proteins.
Fig. 2: Schematic for selective tagging of protein S-nitrosothiols using the SNOTRAP probe.
Fig. 3: Optimization and validation of the SNOTRAP strategy.
Fig. 4: Flash chromatograms of SNOTRAP and intermediate product synthesis.
Fig. 5: SNO-proteomics of 40 post-mortem AD and non-AD human brains as well as 11 post-mortem LBD and non-LBD human brains.

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Data availability

The raw MS data from this study have been deposited into the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifiers PXD020945 and PXD036703.

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Acknowledgements

We gratefully acknowledge the discussion and work of multiple members of the Tannenbaum laboratory at the Massachusetts Institute of Technology and the Lipton laboratory at The Scripps Research Institute, including U. Seneviratne, T. Nakamura, C.-k. Oh and X. Zhang, without whose work the production of this protocol would not have been possible. This work was funded in part by the National Institutes of Health grants (U01 AG088679, R01 AG056259, R35 AG071734, RF1 AG057409, R01 AG056259, R56 AG065372, R01 DA048882, and DP1 DA041722), a California Institute of Regenerative Medicine award (DISC4-16292 ReMIND-L) and the Science and Technology Development Planning Project of Jilin Province in China (grant no. 20240305022YY).

Author information

Authors and Affiliations

  1. Departments of Biological Engineering and Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Hongmei Yang, Haitham Amal & Steven R. Tannenbaum

  2. Public Experimental Center, Changchun University of Chinese Medicine, Changchun, China

    Hongmei Yang

  3. Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel

    Haitham Amal

  4. Rosamund Stone Zander Translational Neuroscience Center, Boston Children’s Hospital, Boston, MA, USA

    Haitham Amal

  5. Department of Neurology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Haitham Amal

  6. Neurodegeneration New Medicines Center and Department of Molecular & Cellular Biology, The Scripps Research Institute, La Jolla, CA, USA

    Stuart A. Lipton

  7. Department of Neurosciences, University of California, San Diego, School of Medicine, La Jolla, CA, USA

    Stuart A. Lipton

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  1. Hongmei Yang
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  2. Haitham Amal
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  3. Steven R. Tannenbaum
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Contributions

Conceptualization: H.Y., S.R.T. and S.A.L. Methodology: H.Y., H.A., S.R.T. and S.A.L. Data collection and analysis: H.Y. and H.A. Investigation: H.Y., H.A., S.R.T. and S.A.L. Writing—original outline: S.A.L.; original draft: H.Y. Writing—reviewing and editing: H.A., S.R.T. and S.A.L. Visualization: H.Y. and S.A.L. Supervision: S.R.T. and S.A.L. Funding acquisition: S.R.T., H.A., H.Y. and S.A.L.

Corresponding authors

Correspondence to Hongmei Yang or Stuart A. Lipton.

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Competing interests

H.A. is the scientific founder of Point6 Bio, a biotechnology company focusing on multiomics discoveries, including S-nitrosylated proteins, for molecular insight into autism spectrum disorder. S.A.L. serves on the scientific advisory board of Point6 Bio. The other authors declare no competing interests.

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Nature Protocols thanks Per Hägglund, Wei-Jun Qian and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references

Yang, H. et al. Sci. Adv. 8, eade0764 (2022): https://doi.org/10.1126/sciadv.ade0764

Andreyev, A. Y. et al. Adv. Sci. 11, 2306469 (2024): https://doi.org/10.1002/advs.202306469

Doulias, P. T. et al. Cell Chem. Biol. 30, 965–975 (2023): https://doi.org/10.1016/j.chembiol.2023.06.018

Yang, H. et al. J. Chromatogr. A 1705, 464162 (2023): https://doi.org/10.1016/j.chroma.2023.464162

Extended data

Extended Data Fig. 1 Photographs of equipment setup for SNOTRAP synthesis.

Photographs of equipment setup during SNOTRAP synthesis. a, Argon balloon and a rubber septum. b, The vacuum pump. c, The argon tank.

Extended Data Fig. 2 UPLC-MS of the SNOTRAP probe.

1H, 13C, and 31P NMR of 2-(diphenylphosphino)-benzenethiol. a, 1H NMR. b, 13C NMR. c, 31P NMR.

Extended Data Fig. 3 1H, 13C, and 31P NMR of 2-(diphenylphosphino)benzenethiol.

UPLC-MS of the SNOTRAP probe. a, Total ion chromatography of 2.5 µm SNOTRAP in methanol. I: oxidized SNOTRAP, II: SNOTRAP. b, Mass spectrum of peak I from panel a in positive ion mode. c, Mass spectrum of peak II from panel a in positive ion mode.

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Yang, H., Amal, H., Tannenbaum, S.R. et al. Proteome-wide profiling of S-nitrosylated proteins using the SNOTRAP probe and mass spectrometry-based detection. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01282-1

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