Ubiquitin Profiling in Liver Using a Transgenic Mouse with Biotinylated Ubiquitin

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.