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Identification of cross talk between SUMOylation and ubiquitylation using a sequential peptide immunopurification approach

Nature Protocols volume 12, pages 2354–2355 (2017)Cite this article

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Abstract

Ubiquitin and ubiquitin-like modifiers (UBLs) such as small ubiquitin-like modifier (SUMO) can act as antagonists to one another by competing to occupy similar residues in the proteome. In addition, SUMO and ubiquitin can be coupled to each other at key lysine residues to form highly branched protein networks. The interplay between these modifications governs important biological processes such as double-strand break repair and meiotic recombination. We recently developed an approach that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation. This protocol requires cells that express a mutant 6×His-SUMO3m protein that has had its C terminus modified from QQQTGG to RNQTGG, enabling the purification of SUMOylated peptides and their identification by tandem mass spectrometry (MS/MS). Cells are lysed under denaturing conditions, and the SUMOylated proteins are purified on nickel-nitrilotriacetic acid (Ni-NTA) resin via the 6×His on the SUMO3m construct. After on-bead digestion using trypsin, ubiquitylated peptides are enriched by immunoprecipitation, and the flow-through from this step is subjected to anti-SUMO immunoprecipitation. The SUMOylated peptides are fractionated on strong cation exchange (SCX) StageTips to enhance the coverage of the SUMO proteome. The ubiquitylated and SUMOylated peptides are analyzed separately by liquid chromatography (LC)–MS/MS and identified with MaxQuant. We demonstrate how this approach can be used to identify temporal changes in SUMOylated and ubiquitylated proteins in response to, for instance, heat shock and proteasome inhibition. The procedure requires 3 d when starting from cell pellets and yields >8,000 SUMO sites and >3,500 ubiquitin sites from 16 mg of cell extract.

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Figure 1: Cross talk between protein SUMOylation and ubiquitylation.
Figure 2: Experimental design to monitor the cross talk between ubiquitin and SUMO from cultured cells.
Figure 3: SUMOylation and ubiquitylation sites can be unambiguously differentiated from each other by MS/MS.
Figure 4: Representative chromatograms for the 6×His-SUMOm remnant diagnostic ions used as quality control for SUMO site identification purposes.
Figure 5: MG132 and heat-shock treatments enhance the coverage of the SUMO proteome and promote the ubiquitylation of SUMOylated proteins.

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References

  1. Bettermann, K., Benesch, M., Weis, S. & Haybaeck, J. SUMOylation in carcinogenesis. Cancer Lett. 316, 113–125 (2012).

    Article  CAS  Google Scholar 

  2. Rodriguez, M.S., Dargemont, C. & Hay, R.T. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276, 12654–12659 (2001).

    Article  CAS  Google Scholar 

  3. Tatham, M.H. et al. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J. Biol. Chem. 276, 35368–35374 (2001).

    Article  CAS  Google Scholar 

  4. Matic, I. et al. In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy. Mol. Cell. Proteomics 7, 132–144 (2008).

    Article  CAS  Google Scholar 

  5. Tatham, M.H., Matic, I., Mann, M. & Hay, R.T. Comparative proteomic analysis identifies a role for SUMO in protein quality control. Sci. Signal. 4, rs4 (2011).

    Article  CAS  Google Scholar 

  6. Geiss-Friedlander, R. & Melchior, F. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 8, 947–956 (2007).

    Article  CAS  Google Scholar 

  7. Bernier-Villamor, V., Sampson, D.A., Matunis, M.J. & Lima, C.D. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108, 345–356 (2002).

    Article  CAS  Google Scholar 

  8. Creton, S. & Jentsch, S. SnapShot: the SUMO system. Cell 143, 848–848.e841 (2010).

    Article  CAS  Google Scholar 

  9. Weisshaar, S.R. et al. Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML. FEBS Lett. 582, 3174–3178 (2008).

    Article  CAS  Google Scholar 

  10. Uzunova, K. et al. Ubiquitin-dependent proteolytic control of SUMO conjugates. J. Biol. Chem. 282, 34167–34175 (2007).

    Article  CAS  Google Scholar 

  11. Geoffroy, M.-C. & Hay, R.T. An additional role for SUMO in ubiquitin-mediated proteolysis. Nat. Rev. Mol. Cell Biol. 10, 564–568 (2009).

    Article  CAS  Google Scholar 

  12. Udeshi, N.D. et al. Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition. Mol. Cell. Proteomics 11, 148–159 (2012).

    Article  CAS  Google Scholar 

  13. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

    Article  CAS  Google Scholar 

  14. Schwertman, P., Bekker-Jensen, S. & Mailand, N. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers. Nat. Rev. Mol. Cell Biol. 17, 379–394 (2016).

    Article  CAS  Google Scholar 

  15. Rao, H.B. et al. A SUMO-ubiquitin relay recruits proteasomes to chromosome axes to regulate meiotic recombination. Science 355, 403–407 (2017).

    Article  Google Scholar 

  16. Hendriks, I.A. et al. Uncovering global SUMOylation signaling networks in a site-specific manner. Nat. Struct. Mol. Biol. 21, 927–936 (2014).

    Article  CAS  Google Scholar 

  17. Lamoliatte, F. et al. Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling. Nat. Commun. 5, 5409 (2014).

    Article  CAS  Google Scholar 

  18. Tammsalu, T. et al. Proteome-wide identification of SUMO2 modification sites. Sci. Signal. 7, rs2 (2014).

    Article  Google Scholar 

  19. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    Article  CAS  Google Scholar 

  20. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  21. Galisson, F. et al. A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells. Mol. Cell. Proteomics 10, M110.004796 (2011).

    Article  Google Scholar 

  22. McManus, F.P., Altamirano, C.D. & Thibault, P. In vitro assay to determine SUMOylation sites on protein substrates. Nat. Protoc. 11, 387–397 (2016).

    Article  CAS  Google Scholar 

  23. Hendriks, I.A. & Vertegaal, A.C. A high-yield double-purification proteomics strategy for the identification of SUMO sites. Nat. Protoc. 11, 1630–1649 (2016).

    Article  CAS  Google Scholar 

  24. Lamoliatte, F. et al. Targeted identification of SUMOylation sites in human proteins using affinity enrichment and paralog-specific reporter ions. Mol. Cell. Proteomics 12, 2536–2550 (2013).

    Article  CAS  Google Scholar 

  25. Lamoliatte, F., McManus, F.P., Maarifi, G., Chelbi-Alix, M.K. & Thibault, P. Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using sequential peptide immunopurification. Nat. Commun. 8, 14109 (2017).

    Article  CAS  Google Scholar 

  26. Udeshi, N.D. et al. Refined preparation and use of anti-diglycine remnant (K-ɛ-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments. Mol. Cell. Proteomics 12, 825–831 (2013).

    Article  CAS  Google Scholar 

  27. Impens, F., Radoshevich, L., Cossart, P. & Ribet, D. Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli. Proc. Natl Acad. Sci. USA 111, 12432–12437 (2014).

    Article  CAS  Google Scholar 

  28. Tammsalu, T. et al. Proteome-wide identification of SUMO modification sites by mass spectrometry. Nat. Protoc. 10, 1374–1388 (2015).

    Article  CAS  Google Scholar 

  29. Barysch, S.V., Dittner, C., Flotho, A., Becker, J. & Melchior, F. Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies. Nat. Protoc. 9, 896–909 (2014).

    Article  CAS  Google Scholar 

  30. Becker, J. et al. Detecting endogenous SUMO targets in mammalian cells and tissues. Nat. Struct. Mol. Biol. 20, 525–531 (2013).

    Article  CAS  Google Scholar 

  31. Hendriks, I.A. & Vertegaal, A.C. A comprehensive compilation of SUMO proteomics. Nat. Rev. Mol. Cell Biol. 17, 581–595 (2016).

    Article  CAS  Google Scholar 

  32. Batth, T.S., Francavilla, C. & Olsen, J.V. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J. Proteome Res. 13, 6176–6186 (2014).

    Article  CAS  Google Scholar 

  33. Svinkina, T. et al. Deep, quantitative coverage of the lysine acetylome using novel anti-acetyl-lysine antibodies and an optimized proteomic workflow. Mol. Cell. Proteomics 14, 2429–2440 (2015).

    Article  CAS  Google Scholar 

  34. Udeshi, N.D., Mertins, P., Svinkina, T. & Carr, S.A. Large-scale identification of ubiquitination sites by mass spectrometry. Nat. Protoc. 8, 1950–1960 (2013).

    Article  CAS  Google Scholar 

  35. Vizcaino, J.A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, 11033 (2016).

    Article  CAS  Google Scholar 

  36. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and Genome Canada. F.P.M. is the recipient of a postdoctoral fellowship from NSERC. F.L. was supported by a scholarship from the faculty of graduate studies of the Université de Montreal. The Institute for Research in Immunology and Cancer (IRIC) receives infrastructure support from Genome Canada and Génome Québec, the Institute for Research in Immunology and Cancer — Commercialization of Research (IRICoR), the Canadian Foundation for Innovation, and the Fonds de Recherche du Québec – Santé (FRQS).

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  1. Francis P McManus and Frédéric Lamoliatte: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Research in Immunology and Cancer, Québec, Canada

    Francis P McManus, Frédéric Lamoliatte & Pierre Thibault

  2. Department of Chemistry,

    Frédéric Lamoliatte & Pierre Thibault

  3. University of Montréal, Québec, Canada

    Frédéric Lamoliatte & Pierre Thibault

  4. Department of Biochemistry,

    Pierre Thibault

  5. University of Montréal, Québec, Canada

    Pierre Thibault

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  1. Francis P McManus
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Contributions

F.P.M. and F.L. carried out the experiments. F.P.M., F.L. and P.T. wrote the manuscript. P.T. developed the concept and managed the project. F.P.M. and F.L. contributed equally to this work.

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Correspondence to Pierre Thibault.

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McManus, F., Lamoliatte, F. & Thibault, P. Identification of cross talk between SUMOylation and ubiquitylation using a sequential peptide immunopurification approach. Nat Protoc 12, 2354–2355 (2017). https://doi.org/10.1038/nprot.2017.105

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