Global profiling of arginine reactivity and ligandability in the human proteome

Global profiling of arginine reactivity and ligandability in the human proteome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Global profiling of arginine reactivity and ligandability in the human proteome Gang Li, Yuena Wang, Shaoshuai Xie, Tao Hu, Lin Zhu, Yansheng Zhai, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5337832/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Nature Chemistry → Version 1 posted You are reading this latest preprint version Abstract Covalent small molecules targeting nucleophilic amino acids have emerged as a rapidly advancing frontier in covalent drug development, with a primary focus on cysteine and lysine residues. Despite the crucial biological roles of arginine, its reactivity and ligandability in the human proteome remain largely unexplored. In this study, we employed the activity-based protein profiling approach using phenylglyoxal-based chemical probes to globally profile arginine residues. By screening a focused library of 13 phenylglyoxal derivatives, we identified probes that offered enhanced coverage and selectivity. This enabled the quantification of 4,606 arginine sites across diverse human cell lines. Among these, two critical arginine residues were found to regulate liquid-liquid phase separation in either cytosol or nucleus. We further assessed arginine reactivity through on-beads reductive dimethylation quantitative proteomics, identifying a subset of hyper-reactive arginines. Competitive fragment screening via DIA-ABPP delineated the ligandability map between arginines and dicarbonyl compounds. This dataset also identified specific ligandable arginines that impact protein activities, revealing potential targets for drug discovery. Moreover, we mapped these ligandable arginines to protein interfaces that exhibited significant changes in solvent accessible surface area upon protein-protein interactions. These findings suggested that ligandable arginines could disrupt these interactions—a hypothesis confirmed through co-immunoprecipitation experiments, which facilitated the discovery of inhibitors for protein-protein interactions. Overall, this study offers a comprehensive profile of arginine reactivity and introduces new insights into the functional and ligandable arginine landscape of the human proteome, underscoring new possibilities of covalent drugs targeting arginines, particularly as inhibitors of protein interactions. Biological sciences/Biochemistry/Proteomics Biological sciences/Biotechnology/Proteomics Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SI20241026WYN.pdf Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Nature Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5337832","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":379563278,"identity":"2a8f5152-0c47-4973-a592-5ca446c35d4a","order_by":0,"name":"Gang 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Despite the crucial biological roles of arginine, its reactivity and ligandability in the human proteome remain largely unexplored. In this study, we employed the activity-based protein profiling approach using phenylglyoxal-based chemical probes to globally profile arginine residues. By screening a focused library of 13 phenylglyoxal derivatives, we identified probes that offered enhanced coverage and selectivity. This enabled the quantification of 4,606 arginine sites across diverse human cell lines. Among these, two critical arginine residues were found to regulate liquid-liquid phase separation in either cytosol or nucleus. We further assessed arginine reactivity through on-beads reductive dimethylation quantitative proteomics, identifying a subset of hyper-reactive arginines. Competitive fragment screening via DIA-ABPP delineated the ligandability map between arginines and dicarbonyl compounds. This dataset also identified specific ligandable arginines that impact protein activities, revealing potential targets for drug discovery. Moreover, we mapped these ligandable arginines to protein interfaces that exhibited significant changes in solvent accessible surface area upon protein-protein interactions. These findings suggested that ligandable arginines could disrupt these interactions—a hypothesis confirmed through co-immunoprecipitation experiments, which facilitated the discovery of inhibitors for protein-protein interactions. 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