Longevity & Aging

PAD2 vs PAD4 Enzyme Selectivity Mapped Across 256 Peptides

Researchers systematically mapped how PAD2 and PAD4 enzymes citrullinate arginine residues by synthesizing 256 combinatorial peptides and analyzing them with advanced mass spectrometry. PAD2 proved broadly active, efficiently modifying 233 of 256 peptides, while PAD4 was far more selective, with flanking amino acids dramatically affecting activity. Proline at the C-terminal position strongly blocked citrullination for both enzymes, while asparagine at either flanking position enhanced it. These findings clarify the distinct substrate rules governing each enzyme and highlight a critical analytical challenge: asparagine-enhanced citrullination can be confused with asparagine deamidation, a common source of false positives in proteomics studies.