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This irreversible post\u2010translational 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\u2010and\u2010mix 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 \u226592% efficiency, with Glu and Pro in the\n                    <jats:italic>C<\/jats:italic>\n                    \u2010terminal position of the target arginine being unfavorable. In contrast, PAD4 showed lower overall conversion, with Pro strongly inhibiting citrullination at the C\u2010terminal position and multiple residues negatively affecting the N\u2010terminal 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.\n                  <\/jats:p>","DOI":"10.1002\/cbic.70408","type":"journal-article","created":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T06:20:39Z","timestamp":1780899639000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Mapping the Substrate Specificity Landscape of PAD2 and PAD4 Enzymes"],"prefix":"10.1002","volume":"27","author":[{"given":"Adina","family":"Borb\u00e9ly","sequence":"first","affiliation":[{"name":"MTA\u2010ELTE Lend\u00fclet (Momentum) Ion Mobility Mass Spectrometry Research Group Department of Analytical Chemistry Institute of Chemistry ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arnold","family":"Steckel","sequence":"additional","affiliation":[{"name":"MTA\u2010ELTE Lend\u00fclet (Momentum) Ion Mobility Mass Spectrometry Research Group Department of Analytical Chemistry Institute of Chemistry ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"D\u00e1vid","family":"Papp","sequence":"additional","affiliation":[{"name":"MTA\u2010ELTE Lend\u00fclet (Momentum) Ion Mobility Mass Spectrometry Research Group Department of Analytical Chemistry Institute of Chemistry ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest Hungary"},{"name":"Hevesy Gy\u00f6rgy PhD School of Chemistry Faculty of Science Institute of Chemistry ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Domonkos","family":"P\u00e1l","sequence":"additional","affiliation":[{"name":"MTA\u2010TTK Lend\u00fclet (Momentum) Glycan Biomarker Research Group HUN\u2010REN Research Centre for Natural Sciences  Budapest Hungary"},{"name":"Doctoral School Semmelweis University  Budapest Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katalin","family":"Uray","sequence":"additional","affiliation":[{"name":"MTA\u2010ELTE Lend\u00fclet (Momentum) Ion Mobility Mass Spectrometry Research Group Department of Analytical Chemistry Institute of Chemistry ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest Hungary"},{"name":"Institute of Chemistry HUN\u2010REN\u2013ELTE Research Group of Peptide Chemistry, ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vikt\u00f3ria Goldschmidt","family":"G\u0151z","sequence":"additional","affiliation":[{"name":"MTA\u2010ELTE Lend\u00fclet (Momentum) Ion Mobility Mass Spectrometry Research Group Department of Analytical Chemistry Institute of Chemistry ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7637-7133","authenticated-orcid":false,"given":"Gitta","family":"Schlosser","sequence":"additional","affiliation":[{"name":"MTA\u2010ELTE Lend\u00fclet (Momentum) Ion Mobility Mass Spectrometry Research Group Department of Analytical Chemistry Institute of Chemistry ELTE E\u00f6tv\u00f6s Lor\u00e1nd University  Budapest 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