Figure 3.

Activity-based profiling of deubiquitinating enzymes and interacting proteins. (a) Ubiquitin fused to an amino-terminal epitope tag (for example hemagglutinin, HA) and a carboxy-terminal reactive group forms a covalent conjugate with deubiquitinating enzymes (DUBs; for details of the generation of these ubiquitin probes, see [57]). (b) The DUB-ubiquitin conjugates can be immunopurified using the HA epitope. Immunopurification under native conditions allows identification of DUBs and their interacting proteins by mass spectrometry (MS). The immunopurified fractions can be further separated by gel electrophoresis, and DUB-ubiquitin conjugates can be detected by anti-HA immunoblotting. Proteins corresponding to HA-reactive bands can be eluted from silver-stained gels (not shown) and the DUBs can be identified by mass spectrometry.