Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin

Benoît Lectez, Rebekka Migotti, So Young Lee, Juanma Ramirez, Naiara Beraza, Bill Mansfield, James D. Sutherland, Maria L. Martinez-Chantar, Gunnar Dittmar, Ugo Mayor*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

27 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3016-3026
Number of pages11
JournalJournal of Proteome Research
Volume13
Issue number6
DOIs
Publication statusPublished - 6 Jun 2014
Externally publishedYes

Keywords

  • in vivo biotinylation
  • liver
  • mouse model
  • proteomics
  • ubiquitin

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