Chemically labeled ThUBD permits rapid and super-sensitive imaging of polyubiquitination signals

• Published in: Analyst (London) 2022-07, Vol.147 (15), p.3434-3443
• Main Authors: Xiao, Weidi, Huang, Shuai, Gao, Yuan, Liu, Tong, Li, Naikang, Sui, Xinying, Wang, Yonghong, Yang, Guan, Chang, Lei, Kang, Xianjiang, Duan, Changzhu, Qin, Weijie, Xu, Ping, Li, Yanchang
• Format: Article
• Language: English
• Subjects: Antibodies; Binding; Domains; Peroxidase; Sensitivity; Signal detection; Substrates
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/d2an00577h

Polyubiquitination signal deliver diverse cellular signal, which have been recognized as a sophisticated ubiquitin code. The perception and transduction of ubiquitination signal depend on the specificity and sensitivity of the ubiquitin-binding domain. Accurate and sensitive detection of polyubiquitination signal is crucial for revealing the dynamic cellular ubiquitin-regulated events. Western blotting (WB) and immunohistochemistry (IHC) are the most widely used biochemical strategies to detect ubiquitination signal on substrates under diverse physiological and pathological conditions. However, anti-ubiquitin antibodies fail to reflect polyubiquitination signal unbiasedly because of their strong preference for K63-linked ubiquitin chains. Herein, we demonstrated that our previously developed tandem hybrid ubiquitin-binding domain (ThUBD) chemically labeled with a reporter group such as horseradish peroxidase (ThUBD-HRP) could significantly improve the robustness and sensitivity of polyubiquitination signal detection. This advanced method was named TUF-WB Plus (TUF-WB + ). The TUF-WB + method significantly increases the sensitivity and accuracy of ubiquitin detection and requires a shorter experimental operation time. Furthermore, it enables the ThUBD-HRP probe to function as a powerful tool for spatial in situ polyubiquitination detection in cells by immunohistochemistry. Our newly developed ThUBD-HRP probe and TUF-WB + method provide a robust and powerful tool for ubiquitination signal detection with hypersensitivity in an unbiased manner. The ThUBD-HRP probe and the consequential developed TUF-WB + method can detect polyubiquitination signal through one-step incubation with hypersensitivity, unbiased detection and a shorter operation time compared with the antibody method.