The following observations confirm the validity of our assay. First, the bona fide ISG15-isopeptidase USP18 displayed reactivity only towards ISG15VS (Figure 2A ), whereas most of the DUBs reacted only with UbVME. An example is shown with CGI-77 ( Figure 1 , 2F), a previously uncharacterized Otubain-homolog. Second, as a negative control, the SUMO protease SENP2 formed an adduct exclusively with SUMO1VME ( Figure 2G ). We found no evidence for any of the DUBs evaluated here to display reactivity toward the SUMO1 probe (not shown). The presence of the reactive group alone is clearly not sufficient for binding to the active-site cysteine of a protease and specificity of a DUB thus depends on the peptide moiety of the probe, containing either ubiquitin, SUMO1 or ISG15. Lastly, all covalent modifications of DUBs by active-site directed probes were blocked by pretreatment of the translated polypeptides with the sulfhydryl alkylating agent N-ethylmaleimide (NEM) (Figure 2 ), confirming the cysteinedependency of adduct formation.