{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T06:24:12Z","timestamp":1786947852505,"version":"build-2736575974"},"reference-count":25,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Chem."],"abstract":"<jats:p>Disulfide-rich peptide therapeutics often exhibit compact cystine knot-like architectures that endow them with high conformational rigidity, proteolytic stability, and target specificity. Yet their intrinsic polymorphism and structural heterogeneity are difficult to resolve with conventional analytical methods, which lack sufficient power to distinguish subtle structural variants within highly constrained peptide frameworks. Here, cyclic ion mobility-mass spectrometry (cIM-MS), combined with accelerated thermal stress testing, was applied to a manufacturing batch of ziconotide and linaclotide under native and thermally stressed conditions. Pronounced conformational heterogeneity was observed for both drugs even under native conditions. Upon thermal stress, notable degradation and structural reorganization occurred, revealing conformational changes that were not adequately captured by conventional methods. The high-resolution separation achieved by cIM-MS enabled detailed mapping of coexisting conformers and their stress-induced transitions. These results demonstrate that cIM-MS provides a spatially resolved analytical platform for interrogating higher-order structural heterogeneity in disulfide-rich peptide therapeutics, extending the capabilities of conventional mass spectrometry and offering a generalizable approach for structural characterization and quality assessment.<\/jats:p>","DOI":"10.3389\/fchem.2026.1913741","type":"journal-article","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T05:25:11Z","timestamp":1786944311000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Resolving conformational polymorphism in disulfide-rich peptide drugs"],"prefix":"10.3389","volume":"14","author":[{"given":"Shaozhou","family":"Zhu","sequence":"first","affiliation":[{"name":"Institute for Chemical Drug Control, National Institute for Food and Drug Control","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifeng","family":"Ge","sequence":"additional","affiliation":[{"name":"College of New Materials and Chemical Engineering, Beijing Institute of Petrochemical Technology","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiwei","family":"Huang","sequence":"additional","affiliation":[{"name":"Institute for Chemical Drug Control, National Institute for Food and Drug Control","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingzhe","family":"Xu","sequence":"additional","affiliation":[{"name":"Institute for Chemical Drug Control, National Institute for Food and Drug Control","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,8,17]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"9477","DOI":"10.1021\/ja960665u","article-title":"Heterogeneity and stability of helical conformations in peptides: crystallographic and NMR studies of a model heptapeptide","volume":"118","author":"Banerjee","year":"1996","journal-title":"J. Am. Soc. Chem."},{"key":"B2","doi-asserted-by":"publisher","first-page":"1261","DOI":"10.1080\/14656566.2018.1494728","article-title":"Linaclotide for the treatment of chronic constipation","volume":"19","author":"Bassotti","year":"2018","journal-title":"Expert. Opin. Pharmacother."},{"key":"B3","doi-asserted-by":"publisher","first-page":"e9713","DOI":"10.1002\/rcm.9713","article-title":"Recent advancements in disulfide bridge characterization: insights from mass spectrometry","volume":"38","author":"Chaturvedi","year":"2024","journal-title":"Rapid. Commun. Mass. Spectrom."},{"key":"B4","doi-asserted-by":"publisher","first-page":"19012","DOI":"10.1039\/D5SC05617A","article-title":"Directing the oxidative folding of disulfide-rich peptides for enhanced engineering and applications","volume":"16","author":"Cheng","year":"2025","journal-title":"Chem. Sci."},{"key":"B5","doi-asserted-by":"publisher","first-page":"101037","DOI":"10.1016\/j.jpha.2024.101037","article-title":"Determination of related substances in disulfide-rich peptide ziconotide by establishing tandem mass spectrometry methods","volume":"14","author":"Cheng","year":"2024","journal-title":"J. Pharm. Anal."},{"key":"B6","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1016\/S0041-0101(00)00160-4","article-title":"The cystine knot motif in toxins and implications for drug design","volume":"39","author":"Craik","year":"2001","journal-title":"Toxicon"},{"key":"B7","doi-asserted-by":"publisher","first-page":"1099","DOI":"10.1007\/s13361-017-1595-1","article-title":"Assigning peptide disulfide linkage pattern among regio-isomers via methoxy addition to disulfide and tandem mass spectrometry","volume":"28","author":"Durand","year":"2017","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"B8","doi-asserted-by":"publisher","first-page":"1934","DOI":"10.1021\/acs.jpclett.9b03591","article-title":"Mosaic cooperativity in slow polypeptide topological isomerization revealed by residue-specific NMR thermodynamic analysis","volume":"11","author":"Fujinami","year":"2020","journal-title":"J. Phys. Chem. Lett."},{"key":"B9","doi-asserted-by":"publisher","first-page":"1204","DOI":"10.1021\/jasms.2c00053","article-title":"Toward rapid aspartic acid isomer localization in therapeutic peptides using cyclic ion mobility mass spectrometry","volume":"33","author":"Gibson","year":"2022","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"B10","doi-asserted-by":"publisher","first-page":"8564","DOI":"10.1021\/acs.analchem.9b01838","article-title":"A cyclic ion mobility-mass spectrometry system","volume":"91","author":"Giles","year":"2019","journal-title":"Anal. Chem."},{"key":"B11","doi-asserted-by":"publisher","first-page":"901","DOI":"10.1021\/cr400031z","article-title":"Multifaceted roles of disulfide bonds. Peptides as therapeutics","volume":"114","author":"G\u00f3ngora-Ben\u00edtez","year":"2014","journal-title":"Chem. Rev."},{"key":"B12","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1002\/jms.1383","article-title":"Ion mobility\u2013mass spectrometry","volume":"43","author":"Kanu","year":"2008","journal-title":"J. Mass. Spectrom."},{"key":"B13","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1038\/nchem.1889","article-title":"The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics","volume":"6","author":"Lanucara","year":"2014","journal-title":"Nat. Chem."},{"key":"B14","doi-asserted-by":"publisher","first-page":"e31105","DOI":"10.1016\/j.heliyon.2024.e31105","article-title":"A comprehensive review on ziconotide","volume":"10","author":"Lin","year":"2024","journal-title":"Heliyon"},{"key":"B15","doi-asserted-by":"publisher","first-page":"12353","DOI":"10.1039\/d1sc02695j","article-title":"Rational generation of lasso peptides based on biosynthetic gene mutations and site-selective chemical modifications","volume":"12","author":"Liu","year":"2021","journal-title":"Chem. Sci."},{"key":"B16","doi-asserted-by":"publisher","first-page":"1081","DOI":"10.2146\/ajhp130575","article-title":"Linaclotide: a novel agent for chronic constipation and irritable bowel syndrome","volume":"71","author":"Love","year":"2014","journal-title":"Am. J. Health. Syst. Pharm."},{"key":"B17","doi-asserted-by":"publisher","first-page":"4995","DOI":"10.1007\/s00216-015-8664-8","article-title":"Applications of ion-mobility mass spectrometry for lipid analysis","volume":"407","author":"Paglia","year":"2015","journal-title":"Anal. Bioanal. Chem."},{"key":"B18","doi-asserted-by":"publisher","first-page":"870","DOI":"10.1016\/j.jasms.2004.02.006","article-title":"Ion mobility-mass spectrometry applied to cyclic peptide analysis: conformational preferences of gramicidin S and linear analogs in the gas phase","volume":"15","author":"Ruotolo","year":"2004","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"B19","doi-asserted-by":"publisher","first-page":"1139","DOI":"10.1038\/nprot.2008.78","article-title":"Ion mobility\u2013mass spectrometry analysis of large protein complexes","volume":"3","author":"Ruotolo","year":"2008","journal-title":"Nat. Protoc."},{"key":"B20","doi-asserted-by":"publisher","first-page":"1569","DOI":"10.1016\/S0140-6736(10)60354-6","article-title":"Ziconotide for treatment of severe chronic pain","volume":"375","author":"Schmidtko","year":"2010","journal-title":"Lancet"},{"key":"B21","doi-asserted-by":"publisher","first-page":"256","DOI":"10.1002\/prot.26426","article-title":"Cyclic cystine knot and its strong implication on the structure and dynamics of cyclotides","volume":"91","author":"Venkatesan","year":"2023","journal-title":"Proteins"},{"key":"B22","doi-asserted-by":"publisher","first-page":"391","DOI":"10.1021\/ac990601c","article-title":"Separation of isomeric peptides using electrospray ionization\/high-resolution ion mobility spectrometry","volume":"72","author":"Wu","year":"2000","journal-title":"Anal. Chem."},{"key":"B23","doi-asserted-by":"publisher","first-page":"1539","DOI":"10.1038\/s41467-022-29210-x","article-title":"De novo design and directed folding of disulfide-bridged peptide heterodimers","volume":"13","author":"Yao","year":"2022","journal-title":"Nat. Commun."},{"key":"B24","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1002\/jms.241","article-title":"Characterizing closely spaced, complex disulfide bond patterns in peptides and proteins by liquid chromatography\/electrospray ionization tandem mass spectrometry","volume":"37","author":"Yen","year":"2002","journal-title":"J. Mass. Spectrom."},{"key":"B25","doi-asserted-by":"publisher","first-page":"2547","DOI":"10.1039\/C6SC05710A","article-title":"Artificial disulfide-rich peptide scaffolds with precisely defined disulfide patterns and a minimized number of isomers","volume":"8","author":"Zheng","year":"2017","journal-title":"Chem. Sci."}],"container-title":["Frontiers in Chemistry"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fchem.2026.1913741\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T05:25:12Z","timestamp":1786944312000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fchem.2026.1913741\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,17]]},"references-count":25,"alternative-id":["10.3389\/fchem.2026.1913741"],"URL":"https:\/\/doi.org\/10.3389\/fchem.2026.1913741","relation":{},"ISSN":["2296-2646"],"issn-type":[{"value":"2296-2646","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,17]]},"article-number":"1913741"}}