title: In vitro assay to determine SUMOylation sites on protein substrates.
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.
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Francis P
authors.initials: FP
authors.lastname: McManus
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Christine Desroches
authors.initials: CD
authors.lastname: Altamirano
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada.|Department of Chemistry, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002853:Chromatography, Liquid; D019295:Computational Biology; D006801:Humans; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D011506:Proteins / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D058207:Sumoylation; D013997:Time Factors
chemical_list: D011506:Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D008239:Lysine
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