{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T17:16:34Z","timestamp":1788455794639,"version":"build-2803163510"},"reference-count":43,"publisher":"American Chemical Society (ACS)","issue":"9","license":[{"start":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Validation of epitopes and paratopes for antibodies and antigens is crucial for understanding antibody binding and is of interest in the pharmaceutical field. Traditionally, high-resolution techniques like X-ray and NMR are used for delineating protein\u2013protein interactions, but they are prone to sample preparation problems and size limitations. Mass spectrometry (MS)-based structural analysis, like H\/D exchange (HDX), covalent labeling, and cross-linking, contributes unique information and advantages relative to traditional methods. As such, it is desirable to compare all three techniques side by side and assess how complementary their results are for defining epitopes on the targets of interest. In this study, we probed interaction sites of epidermal growth factor receptor (EGFR) with cetuximab and zalutumumab using HDX, covalent labeling, and cross-linking techniques. For covalent labeling experiments, sulfo-NHS acetate and DEPC were used as the labeling reagents. In cross-linking experiments, multiple cross-linkers, including BS3, EDC, DSSO, and ADH\/DMTMM, were used for a comprehensive search of protein\u2013protein interactions. The results from cross-linking and covalent labeling were compared to HDX as well as known epitopes. Covalent labeling, cross-linking, and HDX data showed consistent results for EGFR-Cetuximab epitopes. For the EGFR-Zalutumumab complex, no intermolecular cross-links within reasonable distances were identified, but the epitopes observed from covalent labeling agree with HDX. The results are consistent with reported crystal structures. Overall results with DEPC and sulfo-NHS labeling are comparable to HDX with a faster cycle time, making them amenable for rapid screening of many molecules. DEPC labeling has more residue coverage compared to sulfo-NHS acetate but faces unique challenges like faster hydrolysis and more complex modifications. We further extended the use of covalent labeling experiments in a cellular environment with the same model system and with results consistent with purified proteins, making covalent labeling a suitable method to screen epitopes for complex targets and receptors directly from cells.<\/jats:p>","DOI":"10.1021\/jasms.6c00243","type":"journal-article","created":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T13:02:11Z","timestamp":1787403731000},"page":"2215-2227","source":"Crossref","is-referenced-by-count":0,"title":["Evaluating Antigen\u2013Antibody\nInteractions: A\nComparative Study of Higher-Order Structural MS Methods"],"prefix":"10.1021","volume":"37","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-1120-4127","authenticated-orcid":true,"given":"Yiran","family":"Ma","sequence":"first","affiliation":[{"name":"Eli Lilly and Company , , , ,","place":["San Diego, California, United States, 92121"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristin","family":"Hughes","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , , ,","place":["San Diego, California, United States, 92121"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nary","family":"Choe","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , , ,","place":["San Diego, California, United States, 92121"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Bruemmer","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , , ,","place":["Indianapolis, Indiana, United States, 46225"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jesus A.","family":"Gutierrez","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , , ,","place":["Indianapolis, Indiana, United States, 46225"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deepa","family":"Balasubramaniam","sequence":"additional","affiliation":[{"name":"Eli Lilly and Company , , , ,","place":["San Diego, California, United States, 92121"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"issue":"13","key":"2026090311510421600_cit1","doi-asserted-by":"publisher","first-page":"9086","DOI":"10.1021\/acs.analchem.0c01291","article-title":"Epitope and Paratope Mapping of PD-1\/Nivolumab\nby Mass Spectrometry-Based Hydrogen-Deuterium Exchange, Cross-linking,\nand Molecular Docking","volume":"92","author":"Zhang","year":"2020","journal-title":"Anal. Chem."},{"issue":"7","key":"2026090311510421600_cit2","doi-asserted-by":"publisher","first-page":"1303","DOI":"10.1021\/jasms.2c00108","article-title":"Complementary Structural Information\nfor Antibody-Antigen Complexes from Hydrogen Deuterium Exchange and\nCovalent Labeling Mass Spectrometry","volume":"33","author":"Tremblay","year":"2022","journal-title":"J. Am.\nSoc. Mass Spectrom."},{"key":"2026090311510421600_cit3","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1016\/j.ymeth.2018.04.023","article-title":"Contemporary\nhydrogen deuterium exchange\nmass spectrometry","volume":"144","author":"Oganesyan","year":"2018","journal-title":"Methods"},{"key":"2026090311510421600_cit4","doi-asserted-by":"publisher","first-page":"595","DOI":"10.1038\/s41592-019-0459-y","article-title":"Recommendations\nfor performing, interpreting and reporting\nhydrogen deuterium exchange mass spectrometry (HDX-MS) experiments","volume":"16","author":"Masson","year":"2019","journal-title":"Nat. Methods"},{"issue":"6","key":"2026090311510421600_cit5","doi-asserted-by":"publisher","first-page":"1403","DOI":"10.1038\/nprot.2018.037","article-title":"Revealing the architecture\nof protein\ncomplexes by an orthogonal approach combining HDXMS, CXMS, and disulfide\ntrapping","volume":"13","author":"Xiao","year":"2018","journal-title":"Nat. Protoc."},{"issue":"10","key":"2026090311510421600_cit6","doi-asserted-by":"publisher","first-page":"4355","DOI":"10.1021\/acs.chemrev.9b00815","article-title":"Mass Spectrometry-Based\nProtein Footprinting\nfor Higher-Order Structure Analysis: Fundamentals and Applications","volume":"120","author":"Liu","year":"2020","journal-title":"Chem. Rev."},{"issue":"18","key":"2026090311510421600_cit7","doi-asserted-by":"publisher","first-page":"7178","DOI":"10.1021\/acs.analchem.2c05616","article-title":"Membrane Protein\nBinding Interactions Studied in Live Cells via Diethylpyrocarbonate\nCovalent Labeling Mass Spectrometry","volume":"95","author":"Kirsch","year":"2023","journal-title":"Anal. Chem."},{"issue":"3","key":"2026090311510421600_cit8","doi-asserted-by":"publisher","first-page":"1224","DOI":"10.1039\/C0CS00113A","article-title":"Hydrogen exchange mass spectrometry for studying protein\nstructure and dynamics","volume":"40","author":"Konermann","year":"2011","journal-title":"Chem. Soc. Rev."},{"issue":"19","key":"2026090311510421600_cit9","doi-asserted-by":"publisher","first-page":"7870","DOI":"10.1021\/ac901154s","article-title":"Analysis\nof Protein Conformation and Dynamics by Hydrogen\/Deuterium Exchange\nMS","volume":"81","author":"Engen","year":"2009","journal-title":"Anal. Chem."},{"issue":"30","key":"2026090311510421600_cit10","doi-asserted-by":"publisher","DOI":"10.1016\/j.jprot.2020.103793","article-title":"Evaluation of NHS-Acetate\nand DEPC labelling for determination of solvent accessible amino acid\nresidues in protein complexes","volume":"222","author":"Barth","year":"2020","journal-title":"J. Proteomics"},{"issue":"8","key":"2026090311510421600_cit11","doi-asserted-by":"publisher","first-page":"2895","DOI":"10.1021\/ac701999b","article-title":"Protein Surface Mapping Using Diethylpyrocarbonate\nwith Mass Spectrometric Detection","volume":"80","author":"Mendoza","year":"2008","journal-title":"Anal. Chem."},{"issue":"5","key":"2026090311510421600_cit12","doi-asserted-by":"publisher","first-page":"785","DOI":"10.1002\/mas.20203","article-title":"Probing\nprotein structure by amino acid-specific covalent\nlabeling and mass spectrometry","volume":"28","author":"Mendoza","year":"2009","journal-title":"Mass Spectrom.\nRev."},{"issue":"1","key":"2026090311510421600_cit13","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1002\/mas.21667","article-title":"Membrane protein structures and interactions from covalent\nlabeling coupled with mass spectrometry","volume":"41","author":"Pan","year":"2022","journal-title":"Mass\nSpectrom. Rev."},{"issue":"7","key":"2026090311510421600_cit14","doi-asserted-by":"publisher","first-page":"983","DOI":"10.1016\/j.jasms.2006.03.005","article-title":"Analysis\nof Protein-Protein Interaction Surfaces Using a Combination of Efficient\nLysine Acetylation and nanoLC-MALDI-MS\/MS Applied to the E9:Im9 Bacteriotoxin\u2500Immunity\nProtein Complex","volume":"17","author":"Scholten","year":"2006","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"32","key":"2026090311510421600_cit15","doi-asserted-by":"publisher","first-page":"11969","DOI":"10.1074\/jbc.REV119.006218","article-title":"Fast photochemical oxidation of proteins\n(FPOP): A powerful mass spectrometry-based structural proteomics tool","volume":"294","author":"Johnson","year":"2019","journal-title":"J. Biol. Chem."},{"issue":"1","key":"2026090311510421600_cit16","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1021\/jasms.2c00262","article-title":"Diethylpyrocarbonate-Based\nCovalent Labeling Mass Spectrometry of Protein Interactions in a Membrane\nComplex System","volume":"34","author":"Pan","year":"2023","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"11","key":"2026090311510421600_cit17","doi-asserted-by":"publisher","first-page":"1000","DOI":"10.1038\/s41594-018-0147-0","article-title":"Cross-linking mass spectrometry: methods and applications\nin structural, molecular and systems biology","volume":"25","author":"O\u2019Reilly","year":"2018","journal-title":"Nat. Struct. Mol. Biol."},{"issue":"8","key":"2026090311510421600_cit18","doi-asserted-by":"publisher","first-page":"7500","DOI":"10.1021\/acs.chemrev.1c00786","article-title":"Cross-Linking Mass\nSpectrometry for Investigating Protein Conformations and Protein-Protein\nInteractions: A Method for All Seasons","volume":"122","author":"Piersimoni","year":"2022","journal-title":"Chem.\nRev."},{"issue":"24","key":"2026090311510421600_cit19","doi-asserted-by":"publisher","first-page":"5981","DOI":"10.1007\/s00216-020-02700-x","article-title":"Cross-linking\/mass spectrometry at the crossroads","volume":"412","author":"Piersimoni","year":"2020","journal-title":"Anal. Bioanal. 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Chem."},{"key":"2026090311510421600_cit22","doi-asserted-by":"publisher","first-page":"157","DOI":"10.1016\/j.sbi.2017.10.005","article-title":"Combining cryo-electron\nmicroscopy (cryo-EM) and cross-linking\nmass spectrometry (CX-MS) for structural elucidation of large protein\nassemblies","volume":"46","author":"Schmidt","year":"2017","journal-title":"Curr. Opin. Struct. Biol."},{"issue":"1","key":"2026090311510421600_cit23","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.M110.002212","article-title":"Development of a Novel\nCross-linking Strategy for Fast and Accurate\nIdentification of Cross-linked Peptides of Protein Complexes","volume":"10","author":"Kao","year":"2011","journal-title":"Mol. Cell Proteomics"},{"issue":"26","key":"2026090311510421600_cit24","doi-asserted-by":"publisher","first-page":"9455","DOI":"10.1073\/pnas.1320298111","article-title":"Chemical cross-linking\/mass\nspectrometry targeting acidic residues in proteins and protein complexes","volume":"111","author":"Leitner","year":"2014","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090311510421600_cit25","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1016\/j.ijrobp.2003.11.041","article-title":"Review\nof epidermal growth factor receptor biology","volume":"59","author":"Herbst","year":"2004","journal-title":"Int. J. Radiat. Oncol., Biol., Phys."},{"issue":"4","key":"2026090311510421600_cit26","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1016\/j.ccr.2005.03.003","article-title":"Structural basis for inhibition of the epidermal growth\nfactor receptor by cetuximab","volume":"7","author":"Li","year":"2005","journal-title":"Cancer cell"},{"issue":"16","key":"2026090311510421600_cit27","doi-asserted-by":"publisher","first-page":"6109","DOI":"10.1073\/pnas.0709477105","article-title":"The antibody\nzalutumumab inhibits epidermal growth factor receptor signaling by\nlimiting intra- and intermolecular flexibility","volume":"105","author":"van Bueren","year":"2008","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"issue":"2","key":"2026090311510421600_cit28","doi-asserted-by":"publisher","first-page":"330","DOI":"10.1124\/jpet.113.210765","article-title":"Generation and Activity of a Humanized Monoclonal\nAntibody That Selectively Neutralizes the Epidermal Growth Factor\nReceptor Ligands Transforming Growth Factor-\u03b1 and Epiregulin","volume":"349","author":"Beidler","year":"2014","journal-title":"J. Pharmacol. Exp. Ther."},{"issue":"1","key":"2026090311510421600_cit29","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2021.2000348","article-title":"Development of a robust and semi-automated\ntwo-step antibody purification process","volume":"13","author":"Yang","year":"2021","journal-title":"mAbs"},{"issue":"12","key":"2026090311510421600_cit30","doi-asserted-by":"publisher","first-page":"2580","DOI":"10.1007\/s13361-019-02331-2","article-title":"A Decoupled\nAutomation\nPlatform for Hydrogen\/Deuterium Exchange Mass Spectrometry Experiments","volume":"30","author":"Espada","year":"2019","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"5","key":"2026090311510421600_cit31","doi-asserted-by":"publisher","DOI":"10.1016\/j.jlr.2022.100198","article-title":"An anti-ANGPTL3\/8 antibody decreases circulating triglycerides\nby binding to a LPL-inhibitory leucine zipper-like motif","volume":"63","author":"Balasubramaniam","year":"2022","journal-title":"J. Lipid Res."},{"issue":"D1","key":"2026090311510421600_cit32","doi-asserted-by":"publisher","first-page":"D523","DOI":"10.1093\/nar\/gkac1052","article-title":"UniProt: the Universal\nProtein Knowledgebase in 2023","volume":"51","author":"Bateman","year":"2023","journal-title":"Nucleic Acids\nRes."},{"issue":"26","key":"2026090311510421600_cit33","doi-asserted-by":"publisher","first-page":"13152","DOI":"10.1016\/S0021-9258(18)37684-1","article-title":"Epidermal growth factor receptor threonine and serine\nresidues phosphorylated in vivo","volume":"263","author":"Heisermann","year":"1988","journal-title":"J. Biol. Chem."},{"issue":"4","key":"2026090311510421600_cit34","doi-asserted-by":"publisher","first-page":"1155","DOI":"10.1021\/pr050113n","article-title":"Extended Range Proteomic Analysis\n(ERPA): a new and sensitive LC-MS platform for high sequence coverage\nof complex proteins with extensive post-translational modifications-comprehensive\nanalysis of beta-casein and epidermal growth factor receptor (EGFR)","volume":"4","author":"Wu","year":"2005","journal-title":"J. Proteome Res."},{"issue":"6","key":"2026090311510421600_cit35","doi-asserted-by":"publisher","first-page":"747","DOI":"10.1002\/pro.2458","article-title":"Distance restraints\nfrom crosslinking mass spectrometry:\nMining a molecular dynamics simulation database to evaluate lysine-lysine\ndistances","volume":"23","author":"Merkley","year":"2014","journal-title":"Protein Sci."},{"issue":"3","key":"2026090311510421600_cit36","doi-asserted-by":"publisher","first-page":"1049","DOI":"10.1021\/acs.jproteome.3c00816","article-title":"Leveraging Cross-Linking Mass Spectrometry for Modeling\nAntibody\u2013Antigen Complexes","volume":"23","author":"Di Ianni","year":"2024","journal-title":"J. Proteome\nRes."},{"issue":"3","key":"2026090311510421600_cit37","doi-asserted-by":"publisher","first-page":"541","DOI":"10.1016\/S1097-2765(03)00350-2","article-title":"An Open-and-Shut Case?\nRecent Insights\ninto the Activation of EGF\/ErbB Receptors","volume":"12","author":"Burgess","year":"2003","journal-title":"Mol.\nCell"},{"issue":"1","key":"2026090311510421600_cit38","doi-asserted-by":"publisher","first-page":"25","DOI":"10.3892\/ijo_00000125","article-title":"Anti-epidermal growth factor receptor\nmonoclonal antibody cetuximab\ninhibits EGFR\/HER-2 heterodimerization and activation","volume":"34","author":"Patel","year":"2009","journal-title":"Int. J. Oncol."},{"issue":"6","key":"2026090311510421600_cit39","doi-asserted-by":"publisher","DOI":"10.1007\/s00894-016-3000-6","article-title":"pH dependence of ligand-induced human\nepidermal growth factor receptor activation investigated by molecular\ndynamics simulations","volume":"22","author":"Dong","year":"2016","journal-title":"J. Mol. Model."},{"issue":"5","key":"2026090311510421600_cit40","doi-asserted-by":"publisher","first-page":"694","DOI":"10.1002\/jms.1544","article-title":"Chemical cross-linking\nwith NHS esters: a systematic study on amino acid reactivities","volume":"44","author":"Ma\u0308dler","year":"2009","journal-title":"J. Mass Spectrom."},{"key":"2026090311510421600_cit41","doi-asserted-by":"publisher","first-page":"120","DOI":"10.1016\/j.jprot.2013.03.005","article-title":"Quantitative\ncross-linking\/mass spectrometry\nusing isotope-labelled cross-linkers","volume":"88","author":"Fischer","year":"2013","journal-title":"J. Proteomics"},{"issue":"5","key":"2026090311510421600_cit42","doi-asserted-by":"publisher","first-page":"1030","DOI":"10.1021\/jasms.4c00043","article-title":"Proteins Can Withstand\nMore Extensive Labeling while\nProviding Accurate Structural Information in Covalent Labeling-Mass\nSpectrometry","volume":"35","author":"Kirsch","year":"2024","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"3","key":"2026090311510421600_cit43","doi-asserted-by":"publisher","first-page":"658","DOI":"10.1021\/jasms.9b00132","article-title":"Higher-Order\nStructure Influences the Kinetics of Diethylpyrocarbonate Covalent\nLabeling of Proteins","volume":"31","author":"Pan","year":"2020","journal-title":"J. Am. Soc. Mass Spectrom."}],"container-title":["Journal of the American\nSociety for Mass Spectrometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jasms.6c00243","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/9\/2215\/66987203\/jasms.6c00243.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/9\/2215\/66987203\/jasms.6c00243.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T16:25:48Z","timestamp":1788452748000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jamsef\/article\/37\/9\/2215\/5285693\/Evaluating-Antigen-Antibody-Interactions-A"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,21]]},"references-count":43,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,8,21]]},"published-print":{"date-parts":[[2026,9,2]]}},"URL":"https:\/\/doi.org\/10.1021\/jasms.6c00243","relation":{},"ISSN":["1044-0305","1879-1123"],"issn-type":[{"value":"1044-0305","type":"print"},{"value":"1879-1123","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,21]]}}}