title: A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells.
abstract: The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His(6) tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation.
authors.affiliation: CNRS FRE3238, Institut André Lwoff, Villejuif, France.
authors.forename: Frederic
authors.initials: F
authors.lastname: Galisson
authors.forename: Louiza
authors.initials: L
authors.lastname: Mahrouche
authors.forename: Mathieu
authors.initials: M
authors.lastname: Courcelles
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.forename: Sylvain
authors.initials: S
authors.lastname: Meloche
authors.forename: Mounira K
authors.initials: MK
authors.lastname: Chelbi-Alix
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D000595:Amino Acid Sequence; D002843:Chromatin / Q000737:chemistry; D019295:Computational Biology; D004260:DNA Repair; D057809:HEK293 Cells; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D002843:Chromatin; D020543:Proteome; D025841:Small Ubiquitin-Related Modifier Proteins