{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:13:09Z","timestamp":1787029989078,"version":"build-2736575974"},"reference-count":25,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2022,1,25]],"date-time":"2022-01-25T00:00:00Z","timestamp":1643068800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["U19 AI062629"],"award-info":[{"award-number":["U19 AI062629"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 AI41625"],"award-info":[{"award-number":["R01 AI41625"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.g., 5\u201310 min) for the characterization of protein interactions. We recently developed a long-gradient (e.g., 90 min), sub-zero temperature, ultra-high performance liquid chromatography HDX-MS (UPLC-HDX-MS) platform that has significantly increased separation power and limited back-exchange for the analysis of protein samples with high complexity. In this study, we demonstrated the utility of this platform for mapping antibody\u2013antigen epitopes by examining four fully human monoclonal antibodies to anthrax PA. Antibody p1C03, with limited neutralizing activity in vivo, bound to a region on domain 1A of PA. p6C04 and p1A06, with no neutralizing activities, bound to the same helix on domain 3 to prevent oligomerization of PA. We found p6C01 strongly bound to domain 3 on a different helix region. We also identified a secondary epitope for p6C01, which likely leads to the blocking of furin cleavage of PA after p6C01 binding. This novel binding of p6C01 results in highly neutralizing activity. This is the first report of this distinct binding mechanism for a highly neutralizing fully human antibody to anthrax protective antigen. Studying such epitopes can facilitate the development of novel therapeutics against anthrax.<\/jats:p>","DOI":"10.3390\/toxins14020092","type":"journal-article","created":{"date-parts":[[2022,1,25]],"date-time":"2022-01-25T21:07:11Z","timestamp":1643144831000},"page":"92","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Hydrogen-Deuterium Exchange Mass Spectrometry Reveals a Novel Binding Region of a Neutralizing Fully Human Monoclonal Antibody to Anthrax Protective Antigen"],"prefix":"10.3390","volume":"14","author":[{"given":"Mulin","family":"Fang","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhe","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathleen","family":"Norris","sequence":"additional","affiliation":[{"name":"Department of Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9574-7355","authenticated-orcid":false,"given":"Judith A.","family":"James","sequence":"additional","affiliation":[{"name":"Department of Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104, USA"},{"name":"Department of Microbiology and Immunology, Oklahoma University Health Sciences Center, 940 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA"},{"name":"Department of Medicine and Pathology, Oklahoma University Health Sciences Center, 1000 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Si","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenneth","family":"Smith","sequence":"additional","affiliation":[{"name":"Department of Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"914","DOI":"10.1111\/jcmm.12075","article-title":"The sepsis model: An emerging hypothesis for the lethality of inhalation anthrax","volume":"17","author":"Coggeshall","year":"2013","journal-title":"J. Cell. Mol. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1007\/s10254-004-0028-2","article-title":"Anthrax toxins","volume":"152","author":"Mourez","year":"2004","journal-title":"Rev. Physiol. Biochem. Pharmacol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.mib.2004.12.005","article-title":"Interactions between anthrax toxin receptors and protective antigen","volume":"8","author":"Scobie","year":"2005","journal-title":"Curr. Opin. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kintzer, A.F., Sterling, H.J., Tang, I.I., Williams, E.R., and Krantz, B.A. (2010). Anthrax Toxin Receptor Drives Protective Antigen Oligomerization and Stabilizes the Heptameric and Octameric Oligomer by a Similar Mechanism. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0013888"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"10277","DOI":"10.1073\/pnas.89.21.10277","article-title":"Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin","volume":"89","author":"Klimpel","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"979","DOI":"10.3390\/toxins3080979","article-title":"Mechanism of Lethal Toxin Neutralization by a Human Monoclonal Antibody Specific for the PA20 Region of Bacillus anthracis Protective Antigen","volume":"3","author":"Reason","year":"2011","journal-title":"Toxins"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2030","DOI":"10.1128\/IAI.01254-08","article-title":"Frequency and Domain Specificity of Toxin-Neutralizing Paratopes in the Human Antibody Response to Anthrax Vaccine Adsorbed","volume":"77","author":"Reason","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"105","DOI":"10.3233\/HAB-2004-13402","article-title":"Human monoclonal antibodies that neutralize anthrax toxin by inhibiting heptamer assembly","volume":"13","author":"Wang","year":"2005","journal-title":"Hum. Antibodies"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4276","DOI":"10.1016\/j.vaccine.2012.03.002","article-title":"Human monoclonal antibodies generated following vaccination with AVA provide neutralization by blocking furin cleavage but not by preventing oligomerization","volume":"30","author":"Smith","year":"2012","journal-title":"Vaccine"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"6313","DOI":"10.1128\/IAI.72.11.6313-6317.2004","article-title":"Functional Analysis of Bacillus anthracis Protective Antigen by Using Neutralizing Monoclonal Antibodies","volume":"72","author":"Brossier","year":"2004","journal-title":"Infect. Immun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1038\/s41592-019-0459-y","article-title":"Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments","volume":"16","author":"Masson","year":"2019","journal-title":"Nat. Methods"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1224","DOI":"10.1039\/C0CS00113A","article-title":"Hydrogen exchange mass spectrometry for studying protein structure and dynamics","volume":"40","author":"Konermann","year":"2011","journal-title":"Chem. Soc. Rev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1021\/acs.biochem.8b01123","article-title":"Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Mono-clonal Antibodies against Diphtheria Toxin","volume":"58","author":"Zhu","year":"2019","journal-title":"Biochemistry"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1575","DOI":"10.1021\/jasms.0c00431","article-title":"Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bacteri-cidal Activity of Two Anti-NadA Monoclonal Antibodies","volume":"32","author":"Grauslund","year":"2021","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-018-07780-z","article-title":"HDX-MS reveals dysregulated checkpoints that compromise discrimination against self RNA during RIG-I mediated autoimmunity","volume":"9","author":"Zheng","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"17128","DOI":"10.1073\/pnas.1419686111","article-title":"Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody","volume":"111","author":"Malito","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1016\/j.trac.2018.06.008","article-title":"Conformational analysis of complex protein states by hydrogen\/deuterium exchange mass spectrometry (HDX-MS): Challenges and emerging solutions","volume":"106","author":"Trabjerg","year":"2018","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.aca.2020.11.022","article-title":"High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with sub-zero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis","volume":"1143","author":"Fang","year":"2021","journal-title":"Anal. Chim. Acta"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1038\/nprot.2009.3","article-title":"Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen","volume":"4","author":"Smith","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/ncomms6277","article-title":"MS-GF+ makes progress towards a universal database search tool for proteomics","volume":"5","author":"Kim","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2132","DOI":"10.1007\/s13361-012-0476-x","article-title":"Minimizing Back Exchange in the Hydrogen Exchange-Mass Spectrometry Experiment","volume":"23","author":"Walters","year":"2012","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/bs.mie.2015.06.035","article-title":"Hydrogen Exchange Mass Spectrometry","volume":"566","author":"Mayne","year":"2016","journal-title":"Methods Enzymol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1593","DOI":"10.1021\/jasms.0c00465","article-title":"Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization \"Hotspots\" in the SARS-CoV-2 Spike Protein Receptor Binding Domain\/Human ACE2 Interaction","volume":"32","author":"Narang","year":"2021","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1010","DOI":"10.1126\/science.abd0827","article-title":"Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail","volume":"369","author":"Hansen","year":"2020","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"11315","DOI":"10.1021\/acs.analchem.8b01864","article-title":"Epitope Mapping by HDX-MS Elucidates the Surface Coverage of Antigens Associated with High Blocking Efficiency of Antibodies to Birch Pollen Allergen","volume":"90","author":"Zhang","year":"2018","journal-title":"Anal. Chem."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/2\/92\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:07:17Z","timestamp":1760134037000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/2\/92"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,25]]},"references-count":25,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["toxins14020092"],"URL":"https:\/\/doi.org\/10.3390\/toxins14020092","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,25]]}}}