Activity-based profiling of DUBs Figure 1 shows a consensus phylogram based on the alignment of catalytic core sequences of DUBs, including the majority of known human USP homologs. In this tree, the ISG15-protease USP18 clusters close to USP5 (IsoT1) and its isoform USP13 (IsoT3). Previous work had identified USP5 as a protease with affinity for both ubiquitin [27] and ISG15, as shown by its reaction with an electrophilic ISG15 derivative, ISG15-vinyl sulfone (ISG15VS) [28] . To probe for additional ISG15-reactive proteases, we have cloned and expressed a total of 22 human DUB homologs from different clades of this phylogram (indicated with arrows), 17 of which reacted with a ubiquitin-based probe and/or an ISG15based probe (see below). The screen was based on in vitro transcription and translation (IVT) of cloned cDNAs, which affords a rapid method to generate radiochemically pure proteins. This technique allows the generation of DUBs that cannot be readily expressed in bacterial systems, or that are sequestered in subcellular compartments or in multimolecular complexes when expressed in cell lines. To profile for DUB specificity, we used recombinantly expressed ubiquitin, SUMO1 and ISG15, and installed an electrophilic trap at their C-terminus to obtain the active-site probes ubiquitin-vinylmethyl ester (UbVME), SU-MO1VME and ISG15VS, respectively [28] . DUBs generated by IVT were incubated with each of these three probes (Figure 2 ), followed by direct analysis of the reaction mixture by SDS-PAGE. We have determined by X-ray crystallography that probes of this type form a covalent adduct with the catalytic cysteine residue of active DUBs to yield a thioether-linked adduct between enzyme and probe [26] . When unmodified IVT products are run adjacent to samples incubated with these activity-based probes, the adduct is readily detected through a shift in apparent molecular mass. USP2, USP5, USP13 and USP14 reacted with ISG15VS ( Figure 2B , C, D, E).