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Thioether-mediated protein ubiquitination in constructing affinity- and activity-based ubiquitinated protein probes

Nature Protocols volume 20, pages 3239–3269 (2025)Cite this article

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Abstract

Protein ubiquitination, a critical regulatory mechanism and post-translational modification in eukaryotic cells, involves the formation of an isopeptide bond between ubiquitin (Ub) and targeted proteins. Despite extensive investigation into the roles played by protein ubiquitination in various cellular processes, many questions remain to be answered. A major challenge in the biochemical and biophysical characterization of protein ubiquitination, along with its associated pathways and protein players, lies in the generation of ubiquitinated proteins, either in mono- or poly-ubiquitinated forms. Enzymatic and chemical strategies have been reported to address this challenge; however, there are still unmet needs for the facile generation of ubiquitinated proteins in the quantity and homogeneity required to precisely decipher the role of various protein-specific ubiquitination events. In this protocol, we provide the ubiquitin research community with a chemical ubiquitination method enabled by an α-bromoketone-mediated ligation strategy. This method can be readily adapted to generate mono- and poly-ubiquitinated proteins of interest through a cysteine introduced to replace the target lysine, with the native cysteines mutated to serine. Using proliferating cell nuclear antigen (PCNA) as an example, we present herein a detailed protocol for generating di- and tri-Ub PCNA that contains a photo-activatable cross-linker for capturing potential reader proteins. The thioether-mediated protein ligation and purification typically takes 2–3 weeks. An important feature of our ubiquitination strategy is the ability to introduce a Michael-acceptor warhead to the linkage, allowing the generation of activity-based probes for deubiquitinases and ubiquitin-carrying enzymes such as HECT and RBR E3 ubiquitin ligases and E2 enzymes. As such, our method is highly versatile and can be readily adapted to investigate the readers and erasers of many proteins that undergo reversible ubiquitination.

Key points

  • Studying the role of protein ubiquitination requires well-characterized ubiquitinated protein derivatives. This protocol describes a semisynthetic chemical strategy that uses an α-bromoketone non-hydrolyzable linker (NHL) to connect ubiquitin to proteins of interest (POI).

  • An NHL variant containing a Michael acceptor can be used to trap deubiquitinases (DUBs) and ubiquitin-carrying enzymes. This protocol describes how to prepare the NHL and starting materials and perform reactions with POIs and ubiquitin species.

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Fig. 1: Chemical methods for PCNA ubiquitination.
Fig. 2: Synthesis of the diUb building block.
Fig. 3: Generation of pBpa-triUb-PCNA probe.
Fig. 4: TriUb-PCNA photo-activatable probe and cross-linking.
Fig. 5: Scheme for the synthesis of NHL.
Fig. 6: Scheme for the synthesis of MAL.

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Data availability

The data supporting the findings of this work are provided within the paper. The raw data and related files not included in the current paper can be requested from the corresponding author. Source data are provided with this paper.

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Acknowledgements

This work was supported by National Institutes of Health grants R01GM129468, R35GM152011 and R21AG077189 to Z.Z. and National Institute of General Medical Sciences grants P30 GM110758 and P20 GM104316.

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  1. These authors contributed equally: Gregory A. Davidson, Zeinab Moafian, Amanda R. Sensi.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA

    Gregory A. Davidson, Zeinab Moafian, Amanda R. Sensi & Zhihao Zhuang

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  1. Gregory A. Davidson
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  2. Zeinab Moafian
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Contributions

G.A.D. performed the ubiquitin NHL modifications and ligation to PCNA. Z.M. performed the ubiquitin MAL modifications. A.R.S. performed the synthesis of the linker molecules. G.A.D., Z.M., A.R.S. and Z.Z. wrote the paper.

Corresponding author

Correspondence to Zhihao Zhuang.

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Competing interests

Z.Z. has received a patent (US 9,605,297 B2) on the thioether linker-mediated protein ligation method.

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Key references

Li, G. et al. Chem. Commun. 50, 216–218 (2014): https://doi.org/10.1039/c3cc47382a

Yang, K. et al. ChemBioChem 17, 995–998 (2016): https://doi.org/10.1002/cbic.201600042

Gong, P. et al. Chem. Sci. 9, 7859–7865 (2018): https://doi.org/10.1039/c8sc01573b

Paudel, P. et al. Proc. Natl. Acad. Sci. USA 116, 7288–7297 (2019): https://doi.org/10.1073/pnas.1815027116

Shen, S. et al. Nucleic Acids Res. 49, 9374–9388 (2021): https://doi.org/10.1093/nar/gkab646

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Davidson, G.A., Moafian, Z., Sensi, A.R. et al. Thioether-mediated protein ubiquitination in constructing affinity- and activity-based ubiquitinated protein probes. Nat Protoc 20, 3239–3269 (2025). https://doi.org/10.1038/s41596-025-01162-8

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