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In vitro assay to determine SUMOylation sites on protein substrates

Nature Protocols volume 11, pages 387–397 (2016)Cite this article

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Abstract

Protein SUMOylation regulates the activity of a wide range of cellular substrates, and the identification of small ubiquitin-related modifier (SUMO)-modified sites is often required to understand how this modification affects protein function. However, the site-specific identification of modified lysine residues by mass spectrometry (MS) remains challenging because of the dynamic nature of this modification, its low stoichiometry and the relatively large SUMO remnant left on peptide backbones after tryptic digestion. Here we report a versatile method to identify sites and to profile the extent of modification on recombinant proteins from in vitro SUMOylation assays. We define the steps required for sample preparation, and we describe how to perform proper controls and conduct the liquid chromatography-MS (LC-MS) and bioinformatics analyses. Native protein substrates can be used for the assay, although we recommend the use of His-tagged proteins to facilitate removal of contaminants. The procedure was developed for human SUMO paralogs, and it requires <2 d for completion.

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Figure 1: Protein SUMOylation cascade.
Figure 2: Experimental design for the SUMO site identification of target proteins using an in vitro SUMOylation assay.
Figure 3: Extracted ion chromatograms of SUMOylated peptides.
Figure 4: Kinetic profile of in vitro SUMOylation of RanGAP Lys-524 SUMOylation.
Figure 5: MS/MS spectrum of the [M+3H]3+ ion at m/z 701.0100 from the in vitro SUMOylation of HSP27 at Lys198.
Figure 6: Overlap of the SUMO sites identified from in vitro and in vivo experiments.

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Acknowledgements

This work was funded by the Natural Sciences and Engineering Research Council (NSERC) and Genome Canada. F.P.M. is the recipient of postdoctoral fellowship from NSERC. We thank F. Lamoliatte for technical assistance with the MS analysis, and A. Verreault for PCNA and CBX1 recombinant proteins and for the plasmid encoding CHAF1A. The Institute for Research in Immunology and Cancer (IRIC, Université de Montréal, Montréal, Québec, Canada) receives infrastructure support from IRICoR, the Canadian Foundation for Innovation and the Fonds de Recherche du Québec–Santé (FRQS).

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Authors and Affiliations

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

    Francis P McManus, Christine Desroches Altamirano & Pierre Thibault

  2. Department of Chemistry, Université de Montréal, Montréal, Québec, Canada

    Pierre Thibault

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  1. Francis P McManus
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Contributions

F.P.M. and C.D.A. carried out molecular biology and blotting experiments with the SUMO3 variant. F.P.M. prepared and optimized the current protocol and performed the LC-MS/MS experiments and analyzed the data. F.P.M. and P.T. wrote the manuscript. P.T. developed the concept and managed the project.

Corresponding author

Correspondence to Pierre Thibault.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Comparison of in vitro SUMOylation assay for RanGAP using native SUMO3 and SUMO3 (NQTGG) mutant.

(a) Western blot showing the SUMOylation of RanGAP with native and mutant SUMO3 proteins with and without ATP. MS/MS spectra of the Lys-524 SUMOylated RanGAP peptide with (b) SUMO3 (NQTGG) mutant and (c) native SUMO3 proteins. A series of b- and y-type fragment ions corresponding to the tryptic peptide with a modified K-198 residue is observed when using the SUMO3 mutant and facilitated its identification by the search engine Andromeda. In contrast, limited fragmentation of the peptide backbone is noted for the native SUMO3, and the corresponding MS/MS spectrum could not be identified by Andromeda.

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Supplementary Figure 1 and Supplementary Table 1 (PDF 475 kb)

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McManus, F., Altamirano, C. & Thibault, P. In vitro assay to determine SUMOylation sites on protein substrates. Nat Protoc 11, 387–397 (2016). https://doi.org/10.1038/nprot.2016.023

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