title: In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy.
abstract: The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.
authors.affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Ivan
authors.initials: I
authors.lastname: Matic
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
authors.forename: Martijn
authors.initials: M
authors.lastname: van Hagen
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Schimmel
authors.affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Macek
authors.affiliation: Centre for Molecular Medicine, 61 Biopolis Drive (Proteos), Singapore 138673, Singapore.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Ogg
authors.affiliation: Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Tatham
authors.affiliation: Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
authors.affiliation: Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
authors.affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
authors.forename: Alfred C O
authors.initials: ACO
authors.lastname: Vertegaal
mesh_terms: D000595:Amino Acid Sequence; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D025842:SUMO-1 Protein / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002457:Cell Extracts; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010455:Peptides; D011108:Polymers; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins