A high-throughput method for specific, rapid, precise and efficient detection of protein ubiquitination using...

Protein ubiquitination plays a critical role in cellular signaling, and its dysregulation is closely associated with the pathogenesis of numerous prevalent and refractory diseases. However, high-throughput detection of ubiquitination remains challenging due to the limited affinity and linkage bias of existing tools. Here, we employed an unbiased, high-affinity Tandem Hybrid Ubiquitin Binding Domain (ThUBD) fusion protein-previously developed in our laboratory-to coat 96-well plates densely. This approach enabled the establishment of a high-throughput, sensitive, and specific platform for the identification and quantification of ubiquitinated proteins, allowing unbiased and high-affinity capture of proteins modified with all types of ubiquitin chains. Compared with Tandem Ubiquitin Binding Entity (TUBE)-coated plates, our ThUBD-coated platform exhibits a 16-fold wider linear range for capturing polyubiquitinated proteins from complex proteome samples. It facilitates efficient detection and precise quantification of ubiquitination signals, supporting studies on global ubiquitination profiles as well as target-specific ubiquitination status. Furthermore, it offers robust technical support for the development of Proteolysis-Targeting Chimeras (PROTACs).