Global profiling of arginine reactivity and ligandability in the human proteome

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.