Mapping the Substrate Specificity Landscape of PAD2 and PAD4 Enzymes

Pathological citrullination of proteins is implicated in several diseases, including rheumatoid arthritis, neurodegenerative disorders, and cancer. This irreversible post-translational modification is driven by the peptidyl arginine deiminase (PAD) enzyme family. Although the substrate specificity of the key PAD2 and PAD4 isotypes has been studied, the detailed role of individual amino acids from a multidimensional perspective remains unexplored. Here, we designed a combinatorial library of 256 synthetic peptides to comprehensively map PAD2 and PAD4 substrate preferences. Sixteen natural amino acids were examined in positions flanking the target arginine residue using combinatorial peptide mixtures produced by a split-and-mix synthetic strategy. Relative citrullination efficiencies following PAD treatment were analyzed using mass spectrometry, enhanced by cyclic ion mobility separation. PAD4 displayed higher substrate selectivity than PAD2. PAD2 efficiently citrullinated 233 of the 256 peptides, reaching ≥92% efficiency, with Glu and Pro in the C-terminal position of the target arginine being unfavorable. In contrast, PAD4 showed lower overall conversion, with Pro strongly inhibiting citrullination at the C-terminal position and multiple residues negatively affecting the N-terminal position. Notably, Asn at either flanking position enhanced citrullination for both PAD enzymes. These findings highlighted the need to differentiate arginine citrullination from asparagine deamidation for accurate identification.