Development of Diubiquitin‐Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes

Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′‐Boc‐protected 5‐carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high‐throughput manner.