Thioether-mediated protein ubiquitination in constructing affinity- and activity-based ubiquitinated protein probes.

Protein ubiquitination, a critical regulatory mechanism and post-translational modification in eukaryotic cells, involves the formation of an isopeptide bond between ubiquitin (Ub) and targeted proteins. Despite extensive investigation into the roles played by protein ubiquitination in various cellular processes, many questions remain to be answered. A major challenge in the biochemical and biophysical characterization of protein ubiquitination, along with its associated pathways and protein players, lies in the generation of ubiquitinated proteins, either in mono- or poly-ubiquitinated forms. Enzymatic and chemical strategies have been reported to address this challenge; however, there are still unmet needs for the facile generation of ubiquitinated proteins in the quantity and homogeneity required to precisely decipher the role of various protein-specific ubiquitination events. In this protocol, we provide the ubiquitin research community with a chemical ubiquitination method enabled by an α-bromoketone-mediated ligation strategy. This method can be readily adapted to generate mono- and poly-ubiquitinated proteins of interest through a cysteine introduced to replace the target lysine, with the native cysteines mutated to serine. Using proliferating cell nuclear antigen (PCNA) as an example, we present herein a detailed protocol for generating di- and tri-Ub PCNA that contains a photo-activatable cross-linker for capturing potential reader proteins. The thioether-mediated protein ligation and purification typically takes 2-3 weeks. An important feature of our ubiquitination strategy is the ability to introduce a Michael-acceptor warhead to the linkage, allowing the generation of activity-based probes for deubiquitinases and ubiquitin-carrying enzymes such as HECT and RBR E3 ubiquitin ligases and E2 enzymes. As such, our method is highly versatile and can be readily adapted to investigate the readers and erasers of many proteins that undergo reversible ubiquitination.