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As originally described some 40 years ago, protein ubiquitination was thought to serve primarily as a static mark for protein degradation. In the ensuing years, it has become clear that 'ubiquitination' is a structurally diverse and dynamic post-translational modification and is intricately involved in a myriad of signaling pathways in all eukaryote cells. And like other key pathways in the functional proteome, ubiquitin signaling is often disrupted, sometimes severely so, in human pathophysiology. As a result of its central role in normal physiology and human disease, the ubiquitination field is now represented across the full landscape of biomedical research from fundamental structural and biochemical studies to translational and clinical research. In recent years, mass spectrometry has emerged as a powerful technology for the detection and characterization of protein ubiquitination. Herein we detail qualitative and quantitative proteomic methods using a compare/contrast approach to highlight their strengths and weaknesses., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jarrod A Marto reports financial support was provided by Vertex Pharmaceuticals Incorporated. Jarrod A Marto reports financial support was provided by AstraZeneca Pharmaceuticals LP. Jarrod A Marto reports financial support was provided by Taiho Oncology Inc. Jarrod A Marto reports financial support was provided by SpringWorks Therapeutics Inc. Jarrod A Marto reports financial support was provided by TUO Therapeutics. Sara J Buhrlage reports financial support was provided by AbbVie Inc. Sara J Buhrlage reports financial support was provided by TUO Therapeutics. Sara J Buhrlage reports equipment, drugs, or supplies was provided by AbbVie Inc. Sara J Buhrlage reports equipment, drugs, or supplies was provided by Novartis Institutes for BioMedical Research Inc., (Copyright © 2023. Published by Elsevier B.V.)

Covalent drugs constitute cornerstones of modern medicine. The past decade has witnessed growing enthusiasm for development of covalent inhibitors, fueled by clinical successes as well as advances in analytical techniques associated with the drug discovery pipeline. Among these, mass spectrometry-based chemoproteomic methods stand out due to their broad applicability from focused analysis of electrophile-containing compounds to surveying proteome-wide inhibitor targets. Here, we review applications of both foundational and cutting-edge chemoproteomic techniques across target identification, hit discovery, and lead characterization/optimization in covalent drug discovery. We focus on the practical aspects necessary for the general drug discovery scientist to design, interpret, and evaluate chemoproteomic experiments. We also present three case studies on clinical stage molecules to further showcase the real world significance and future opportunities of these methodologies.

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents., Competing Interests: Declaration of Interests E.S.F. is a founder, advisor, and equity holder in Civetta Therapeutics and Jengu Therapeutics. E.S.F. is an equity holder in C4 Therapeutics, and a consultant to Astellas, Novartis, Deerfield, and EcoR1 capital. The Fischer lab receives or has received research funding from Novartis,Astellas, and Deerfield., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.