Protein ubiquitination is a multi-functional post-translational modification affecting all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation 1-3 . Individual ubiquitin modifications generating the ubiquitin code have been mapped through advances in mass spectrometry, but the architecture of polyubiquitin signals has remained largely inaccessible.We here introduce Ub-clipping as a methodology to understand polyubiquitin signals and architectures. Ub-clipping utilises an engineered viral protease, Lb pro *, that removes ubiquitin incompletely from substrates, such that the signature C-terminal GlyGly dipeptide remains attached to the modified residue, simplifying direct assessment of protein ubiquitination on *

SignificanceAn understanding of the mechanisms by which viruses evade host immunity is essential to the development of antiviral drugs and viral detection strategies. Ubiquitin and ubiquitin-like modifications are crucial in cellular innate immune and infection responses and are often suppressed by viral proteins. We here identify a previously unknown mechanism of viral evasion. A viral protease, Lbpro, removes ubiquitin and the ubiquitin-like protein ISG15 incompletely from proteins. While this strategy efficiently and irreversibly shuts down these modification systems, it enables repurposing of tools and technologies developed for ubiquitin research in virus detection. Specifically, we show that foot-and-mouth disease virus infection can be detected using an anti-GlyGly antibody developed for ubiquitin mass spectrometry research.

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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.