Ub-clipping: an approach for studying ubiquitin chain architecture

Abstract

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 chains 1-3 . 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, Lb pro *, 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.the architecture of ubiquitin signals on substrates (i.e. ubiquitinated proteins or polyubiquitin chains) including: 1) in vitro assembly reactions, 2) total cell lysates, 3) polyubiquitin chains enriched from cells, and 4) isolated damaged mitochondria. The starting point for this protocol is the expression and purification of Lb pro *, which routinely yields large quantities of highly pure and active enzyme. After purification, ubiquitinated substrates are digested with Lb pro *. This results in the collapse of polyubiquitin chains to mono-ubiquitin species. These mono-ubiquitin species consist of ubiquitin moieties with a 'clipped' C-terminus and ubiquitin molecules with one or more GlyGlymodifications (+114 Da). These mass differences, plus any additional chemical modifications (e.g. 4 phosphorylation or acetylation) are easily distinguishable by mass and can be quantified using standard intact mass spectrometry methods and software. In the last sections of this protocol, we describe procedures for the preparation of ubiquitin samples for Ub-clipping and then discuss the analysis of these samples by mass spectrometry.

Quantitative Middle-Down MS Analysis of Parkin-Mediated Ubiquitin Chain Assembly

“…Previous work has shown that Parkin is unable to incorporate pSer65-Ub into chains when pSer65-Ub is the only source of Ub. 34,44 Our middle-down data reveal that subunits within a chain are phosphorylated when PINK1 can act on Ub anytime during the chain assembly process. To determine whether phosphorylation occurs on a particular chain type, we performed electron transfer dissociation (ETD) MS/MS analysis on the diGly-pSer65-Ub 1-74 species.…”

A versatile new ubiquitin detection and purification tool derived from a bacterial deubiquitylase

“…This will greatly increase the percentage of di-Gly-linked peptides present in the digested sample. Since OtUBD has exceptionally high affinity towards free ubiquitin, it could also be used with the Ub-Clipping technique (77). In Ub-Clipping, ubiquitylated proteins are cleaved at the ubiquitylation sites by the protease Lb-Pro to generate diGly-linked monoubiquitin species and free ubiquitin 1-74 .…”