biostudies-literatureYoshida YJapan Society for the Promotion of Science4630-5https://www.ebi.ac.uk/biostudies/studies/S-EPMC4403176biostudies-literatureUnknown112(15)The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrateProceedings of the National Academy of Sciences of the United States of AmericaA comprehensive method for detecting ubiquitinated substrates using TR-TUBE.PMC440317624580152261137713J078522411200821000012Shindo MSaeki YKawawaki JMurakami AYoshihara HYoshida YTanaka KTsuchiya HfalseA comprehensive method for detecting ubiquitinated substrates using TR-TUBE.The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrate2015-01-01T00:00:00Z2015 Apr2026-04-13T08:26:10.177Z2026-04-07T13:29:03.365ZS-EPMC44031762582722710.1073/pnas.1422313112