Acyl azide modification of the ubiquitin C-terminus enables DUB capture

Deubiquitinating enzyme (DUB) abnormalities are associated with many diseases. Previous attempts have been made to introduce various chemical groups such as alkynes, unsaturated olefins and alkyl halides to the C-terminus of ubiquitin (Ub) to capture the active-site cysteine residue in DUBs for structural and biochemical studies. Here, we find that a Ub C-terminal acyl azide can capture DUBs, thereby forming thioester bonds in buffers and cell lysates. This finding not only makes ubiquitin acyl azide a chemical probe for capturing DUBs, but also extends the utility of azide groups in biological applications.