title: System-wide identification of wild-type SUMO-2 conjugation sites.
abstract: SUMOylation is a reversible post-translational modification (PTM) regulating all nuclear processes. Identification of SUMOylation sites by mass spectrometry (MS) has been hampered by bulky tryptic fragments, which thus far necessitated the use of mutated SUMO. Here we present a SUMO-specific protease-based methodology which circumvents this problem, dubbed Protease-Reliant Identification of SUMO Modification (PRISM). PRISM allows for detection of SUMOylated proteins as well as identification of specific sites of SUMOylation while using wild-type SUMO. The method is generic and could be widely applied to study lysine PTMs. We employ PRISM in combination with high-resolution MS to identify SUMOylation sites from HeLa cells under standard growth conditions and in response to heat shock. We identified 751 wild-type SUMOylation sites on endogenous proteins, including 200 dynamic SUMO sites in response to heat shock. Thus, we have developed a method capable of quantitatively studying wild-type mammalian SUMO at the site-specific and system-wide level.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.
authors.forename: Ivo A
authors.initials: IA
authors.lastname: Hendriks
authors.affiliation: Department for Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Rochelle C
authors.initials: RC
authors.lastname: D'Souza
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.
authors.forename: Jer-Gung
authors.initials: JG
authors.lastname: Chang
authors.affiliation: Department for Proteomics and Signal Transduction, Max Planck Institute for 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, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.
authors.forename: Alfred C O
authors.initials: AC
authors.lastname: Vertegaal
mesh_terms: D002853:Chromatography, Liquid; D004586:Electrophoresis; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D058207:Sumoylation; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins