title: Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin.
abstract: Ubiquitination is behind most cellular processes, with ubiquitin substrates being regulated variously according to the number of covalently conjugated ubiquitin molecules and type of chain formed. Here we report the first mammalian system for ubiquitin proteomics allowing direct validation of the MS-identified proteins. We created a transgenic mouse expressing biotinylated ubiquitin and demonstrate its use for the isolation of ubiquitinated proteins from liver and other tissues. The specificity and strength of the biotin-avidin interaction allow very stringent washes, so only proteins conjugated to ubiquitin are isolated. In contrast with recently available antibody-based approaches, our strategy allows direct validation by immunoblotting, therefore revealing the type of ubiquitin chains (mono or poly) formed in vivo. We also identify the conjugating E2 enzymes that are ubiquitin-loaded in the mouse tissue. Furthermore, our strategy allows the identification of candidate cysteine-ubiquitinated proteins, providing a strategy to identify those on a proteomic scale. The novel in vivo system described here allows broad access to tissue-specific ubiquitomes and can be combined with established mouse disease models to investigate ubiquitin-dependent therapeutical approaches.
authors.affiliation: CIC bioGUNE, Bizkaia Teknologia Parkea , Building 801-A, 48160 Derio, Basque Country, Spain.
authors.forename: Benoît
authors.initials: B
authors.lastname: Lectez
authors.forename: Rebekka
authors.initials: R
authors.lastname: Migotti
authors.forename: So Young
authors.initials: SY
authors.lastname: Lee
authors.forename: Juanma
authors.initials: J
authors.lastname: Ramirez
authors.forename: Naiara
authors.initials: N
authors.lastname: Beraza
authors.forename: Bill
authors.initials: B
authors.lastname: Mansfield
authors.forename: James D
authors.initials: JD
authors.lastname: Sutherland
authors.forename: Maria L
authors.initials: ML
authors.lastname: Martinez-Chantar
authors.forename: Gunnar
authors.initials: G
authors.lastname: Dittmar
authors.forename: Ugo
authors.initials: U
authors.lastname: Mayor
mesh_terms: D000818:Animals; D019921:Biotinylation; D008099:Liver / Q000378:metabolism*; D008297:Male; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D020543:Proteome / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D020543:Proteome; D025801:Ubiquitin; D057149:Ubiquitinated Proteins