title: Detecting endogenous SUMO targets in mammalian cells and tissues.
abstract: SUMOylation is an essential modification that regulates hundreds of proteins in eukaryotic cells. Owing to its dynamic nature and low steady-state levels, endogenous SUMOylation is challenging to detect. Here, we present a method that allows efficient enrichment and identification of endogenous targets of SUMO1 and the nearly identical SUMO2 and 3 (SUMO 2/3) from vertebrate cells and complex organ tissue. Using monoclonal antibodies for which we mapped the epitope, we enriched SUMOylated proteins by immunoprecipitation and peptide elution. We used this approach in combination with MS to identify SUMOylated proteins, which resulted in the first direct comparison of the endogenous SUMO1- and SUMO2/3-modified proteome in mammalian cells, to our knowledge. This protocol provides an affordable and feasible tool to investigate endogenous SUMOylation in primary cells, tissues and organs, and it will facilitate understanding of SUMO's role in physiology and disease.
authors.affiliation: Zentrum für Molekulare Biologie Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Becker
authors.forename: Sina V
authors.initials: SV
authors.lastname: Barysch
authors.forename: Samir
authors.initials: S
authors.lastname: Karaca
authors.forename: Claudia
authors.initials: C
authors.lastname: Dittner
authors.forename: He-Hsuan
authors.initials: HH
authors.lastname: Hsiao
authors.forename: Mauricio
authors.initials: M
authors.lastname: Berriel Diaz
authors.forename: Stephan
authors.initials: S
authors.lastname: Herzig
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Frauke
authors.initials: F
authors.lastname: Melchior
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D025841:Small Ubiquitin-Related Modifier Proteins