Decoding isozyme-specific substrate recognition in protein arginine deiminases by in vitro lysate-based citrullination mapping

Abstract Protein arginine deiminases (PADs) convert arginine to citrulline, altering protein structure. Of the five human isozymes, PAD1–4 are catalytically active with distinct tissue-specificities, yet isozyme-specific substrate recognition remains poorly defined. Here, we perform in vitro lysate-based substrate profiling of PAD1–4 via mass spectrometry across human H4 and HeLa cell lines, identifying ~30,000 citrullination sites across ~5,500 proteins. Only 14% of sites are shared among all, reflecting distinct sequence preferences: PAD1–2 show broad specificity, whereas PAD3–4 favor arginines flanked by acidic or glycine residues. These preferences persist over 10 min–16 h, indicating sequence context rather than temporal dynamics drives specificity. Mutation analysis of eleven PAD4 variants reveal Q346, G403, R639, and H640 as key determinants distinguishing substrate recognition from that of PAD2. This work provides a comprehensive in vitro atlas of maximal substrate capacity, defining isozyme-specific motifs and molecular determinants to guide selective inhibitors and probes for citrullination mechanisms in health and disease.