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

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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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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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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