While recombinant USP14 in its purified form bound to electrophilic probes ( Figure 3C ), we did not detect robust hydrolytic activity against ubiquitin-AMC or against ubiquitinor ISG15-linked isopeptide fusion proteins (data not shown). However, using sequential ultracentrifugation to obtain a cytosolic fraction that is enriched in 26S proteasomes [29] , we could show that proteases in this fraction efficiently and specifically cleave an ISG15-isopeptide linked substrate ( Figure 5A , B). The absence of proteolytic intermediates suggests specific cleavage of the isopeptide bond. In addition, the same bait peptide linked to SUMO1 was stable and not hydrolyzed, even upon prolonged incubation for over 24 hours with the proteasome fraction. Proteolysis of the ISG15-linked peptide substrate was inhibited by inclusion of NEM, indicating cleavage by cysteine proteases. Analysis by reaction with ISG15VS supports that USP14 is the only active Red arrows depict DUBs that bound to neither probe (UbVME, ISG15VS, or SUMO1VS), whereas black arrows indicate DUBs that formed covalent adducts with the indicated probes. Our screen represents the first biochemical proof for protease activity of USP13 and the Otubainhomolog CGI-77 (DUB homologs without publication record regarding biochemical function are marked with an asterisk). Otubain1 (OTU1) is an exception in that it binds to alkylhalide-or aldehyde-based probes, but not to the Michael acceptors employed in this study (data not shown). doi:10.1371/journal.pone.0000679.g001 ISG15-specific protease in the proteasome-enriched fraction ( Figure 5C ). While we cannot formally exclude the possibility of a yet undefined enzyme binding to ISG15VS, we consider this unlikely: such a protease would have to display a mass highly similar to that of USP14 and, furthermore, it would have to sediment after centrifugation for 5 hours at 100,000 g. However, only few deubiquitinating enzymes are sedimentable, none at a level comparable to USP14 [29, 31] .