Ub-clipping: an approach for studying ubiquitin chain architecture

The post-translational modification of proteins with ubiquitin is a dynamic multifaceted process affecting all aspects of eukaryotic cellular biology. The complexity of ubiquitin modifications arises from their ability to form architecturally distinct polyubiquitin chains1-3. Despite our understanding of the importance of these signals, we currently lack tools and methods to study them. Here we describe an approach, termed Ub-clipping, which provides unprecedented insight into ubiquitin chain architecture. This protocol is related to our recent Nature paper titled, “Insights into ubiquitin chain architecture using Ub-clipping”. This technology takes advantage of an engineered viral protease, Lbpro*, which ‘clips’ ubiquitin such that the information on the site of modification is retained and the remaining ubiquitin and substrate polypeptides are kept intact. The goal of this protocol is to allow researchers to efficiently adapt our new technology to their proteomic workflows. We anticipate this method will continue to shed light on the architecture of ubiquitin signals, and therefore further our understanding of the ubiquitin code across a broad spectrum of biological systems.