Quantitative Reactivity Profiling of Functional Arginine Residues in Human Cancer Cell Line Proteomes

Abstract Arginine, a critical amino acid for protein structure and function, is involved in enzyme catalysis and macromolecular interactions. However, selectively targeting its reactive guanidine group has been challenging. Here, we utilized a probe, AP‐1 , based on phenylglyoxal, which demonstrated remarkable chemical selectivity and reactivity toward arginine residues. Using activity‐based protein profiling (ABPP), we explored the human proteome across four cancer cell lines, obtaining quantitative data for approximately 17 000 arginine residues. This analysis led to the identification of several previously unreported hyperreactive arginine residues, including R43 of PKM, R171 of LDHA, R172 of LDHB, R341 of CKB, R168 of EIF4A1, and R118 of FUBP1, which are crucial for protein function. Notably, the mutation of CKB's R341 inhibited cell proliferation and migration by downregulating energy supply. We also introduced ArGO‐LDHA‐1 , a covalent inhibitor targeting LDHA's hyperreactive arginine residues, showing potential to enhance chemotherapy efficacy. This work highlights the biological significance of arginine residues and provides a platform for large‐scale profiling of arginine reactivity.