{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T06:51:51Z","timestamp":1793083911102,"version":"4.5.0"},"reference-count":30,"publisher":"American Chemical Society (ACS)","issue":"40","license":[{"start":{"date-parts":[[2027,9,28]],"date-time":"2027-09-28T00:00:00Z","timestamp":1822089600000},"content-version":"vor","delay-in-days":365,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T00:00:00Z","timestamp":1790553600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T00:00:00Z","timestamp":1790553600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T00:00:00Z","timestamp":1790553600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100015015","name":"Government of South Australia","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100015015","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001786","name":"University of Adelaide","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001786","id-type":"DOI","asserted-by":"publisher"}]},{"name":"CSL Innovation Pty Ltd"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Characterizing post-translational modifications (PTMs) in monoclonal antibody products is critical for ensuring product quality attributes understanding and monitoring. While the bottom-up approach is widely adopted, it suffers from the loss of molecular connectivity and is susceptible to artifact generation. Conversely, top-down approaches face limitations due to the high complexity and poor gas-phase fragmentation of large intact molecules to get site-specific post-translational information profiling. Middle-down MS methods offer a complementary middle-ground approach to both bottom-up and top-down, with broader sequence coverage, and facilitate the simultaneous identification of multiple attributes and proteoforms. In this study, we optimized the electron-activated dissociation (EAD) approach for middle-down sequencing of NISTmAb subunits and present a potential MS and MS\/MS strategy to rapidly characterize PTMs on antibodies, using methionine oxidation as an example. Peroxide-stressed and control mAb samples were digested and reduced into subunits using the IdeS enzyme and analyzed via LC-MS, with the optimized MRMHR EAD method. The resulting data enabled high-resolution subunit mass analysis, sequence confirmation, and potential for site-specific oxidation localization. Methionine oxidation localization in NISTmAb was achieved in light chain (M4), Fd\u2019 (M34, M101), and Fc\/2 (M16, M122, M192), with these locations confirmed with bottom-up peptide mapping data. Quantification using the Fc\/2 subunit enabled detection of oxidation levels up to 1% relative, with strong correlation (R2 &amp;gt; 0.99) between expected and observed values using MS1 data. This middle-down electron-activated dissociation (EAD) approach provides a potential method for site-specific characterization of PTMs in antibody products, offering valuable complementary mapping to support formulation development and production.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c03432","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T01:02:07Z","timestamp":1790643727000},"page":"29262-29272","source":"Crossref","is-referenced-by-count":1,"title":["Middle-Down Mass Spectrometry Characterization of\nMonoclonal Antibody Enabled by Electron-Activated Dissociation on\na Time-of-Flight Platform: A Case Study of Methionine Oxidation PTM\nIdentification and Quantification"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8225-145X","authenticated-orcid":true,"given":"Abdulafeez","family":"Akinloye","sequence":"first","affiliation":[{"name":"Adelaide University , , , ,","place":["Adelaide, South Australia, Australia, 5000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leigh","family":"Donnellan","sequence":"additional","affiliation":[{"name":"Adelaide University , , , ,","place":["Adelaide, South Australia, Australia, 5000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0139-9757","authenticated-orcid":true,"given":"Parul","family":"Mittal","sequence":"additional","affiliation":[{"name":"Adelaide University , , , ,","place":["Adelaide, South Australia, Australia, 5000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clifford","family":"Young","sequence":"additional","affiliation":[{"name":"Adelaide University , , , ,","place":["Adelaide, South Australia, Australia, 5000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariam","family":"Nassiri","sequence":"additional","affiliation":[{"name":"CSL Innovation , , ,","place":["Melbourne, Victoria, Australia, 3000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1375-9820","authenticated-orcid":true,"given":"Mark R.","family":"Condina","sequence":"additional","affiliation":[{"name":"Adelaide University , , , ,","place":["Adelaide, South Australia, Australia, 5000"]},{"name":"Mass Dynamics , , ,","place":["Melbourne, Victoria, Australia, 3000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan","family":"Edwards","sequence":"additional","affiliation":[{"name":"CSL Innovation , , ,","place":["Melbourne, Victoria, Australia, 3000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alok","family":"Shah","sequence":"additional","affiliation":[{"name":"CSL Innovation , , ,","place":["Melbourne, Victoria, Australia, 3000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Hoffmann","sequence":"additional","affiliation":[{"name":"Adelaide University , , , ,","place":["Adelaide, South Australia, Australia, 5000"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,28]]},"reference":[{"issue":"1","key":"2026101308064240200_cit1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2024.2443538","article-title":"Antibodies to watch\nin 2025","volume":"17","author":"Crescioli","year":"2025","journal-title":"mAbs"},{"key":"2026101308064240200_cit2","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1016\/j.copbio.2019.01.014","article-title":"Chemical and physical instabilities\nin manufacturing and storage\nof therapeutic proteins","volume":"60","author":"Krause","year":"2019","journal-title":"Curr. Opin. Biotechnol."},{"issue":"1","key":"2026101308064240200_cit3","doi-asserted-by":"publisher","first-page":"169","DOI":"10.1016\/j.xphs.2019.08.009","article-title":"Physicochemical Stability of Monoclonal Antibodies:\nA Review","volume":"109","author":"Le Basle","year":"2020","journal-title":"J. Pharm. Sci."},{"issue":"1","key":"2026101308064240200_cit4","doi-asserted-by":"publisher","DOI":"10.1007\/s11095-018-2545-8","article-title":"Impact of Tryptophan\nOxidation in Complementarity-Determining Regions\nof Two Monoclonal Antibodies on Structure-Function Characterized by\nHydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon\nResonance","volume":"36","author":"Hageman","year":"2019","journal-title":"Pharm. Res."},{"issue":"15","key":"2026101308064240200_cit5","doi-asserted-by":"publisher","first-page":"7536","DOI":"10.1021\/ac501300m","article-title":"Detection\nof Histidine Oxidation in a Monoclonal Immunoglobulin Gamma (IgG)\n1 Antibody","volume":"86","author":"Amano","year":"2014","journal-title":"Anal. Chem."},{"issue":"4","key":"2026101308064240200_cit6","doi-asserted-by":"publisher","first-page":"347","DOI":"10.3109\/10715762.2015.1007968","article-title":"Exploring oxidative\nmodifications of tyrosine: An update on mechanisms of formation, advances\nin analysis and biological consequences","volume":"49","author":"Houe\u0301e-Le\u0301vin","year":"2015","journal-title":"Free\nRadical Res."},{"issue":"3\u20134","key":"2026101308064240200_cit7","doi-asserted-by":"publisher","first-page":"154","DOI":"10.33892\/aph.2021.91.154-155","article-title":"Is the Multi-Attribute Method (MAM)\nthe Next Big Thing?\nA High-Resolution Accurate Mass Multi-Attribute Method for Critical\nQuality Attribute Monitoring","volume":"91","author":"Ko\u0301re\u0301h","year":"2021","journal-title":"Acta Pharm. Hung."},{"issue":"1","key":"2026101308064240200_cit8","doi-asserted-by":"publisher","first-page":"12","DOI":"10.1016\/j.ab.2009.05.018","article-title":"An improved trypsin digestion method minimizes\ndigestion-induced modifications on proteins","volume":"392","author":"Ren","year":"2009","journal-title":"Anal. Biochem."},{"issue":"4","key":"2026101308064240200_cit9","doi-asserted-by":"publisher","first-page":"463","DOI":"10.1016\/j.tibtech.2021.08.006","article-title":"Progress and challenges\nin mass spectrometry-based\nanalysis of antibody repertoires","volume":"40","author":"Snapkov","year":"2022","journal-title":"Trends Biotechnol."},{"issue":"7","key":"2026101308064240200_cit10","doi-asserted-by":"publisher","DOI":"10.1016\/j.mcpro.2025.100989","article-title":"Top-Down and Middle-Down Mass Spectrometry of Antibodies","volume":"24","author":"Khristenko","year":"2025","journal-title":"Mol. Cell. 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Mass Spectrom."},{"issue":"13","key":"2026101308064240200_cit13","doi-asserted-by":"publisher","first-page":"8827","DOI":"10.1021\/acs.analchem.0c00293","article-title":"Middle Level IM\u2013MS\nand CIU Experiments for Improved Therapeutic Immunoglobulin Subclass\nFingerprinting","volume":"92","author":"Botzanowski","year":"2020","journal-title":"Anal. Chem."},{"issue":"2","key":"2026101308064240200_cit14","doi-asserted-by":"publisher","first-page":"264","DOI":"10.1021\/jasms.2c00281","article-title":"Novel Hybrid Quadrupole-Multireflecting Time-of-Flight\nMass Spectrometry System","volume":"34","author":"Cooper-Shepherd","year":"2023","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"4","key":"2026101308064240200_cit15","doi-asserted-by":"publisher","first-page":"581","DOI":"10.1002\/mas.21902","article-title":"Recent advances in high-resolution traveling wave-based ion mobility\nseparations coupled to mass spectrometry","volume":"44","author":"Naylor","year":"2025","journal-title":"Mass\nSpectrom. Rev."},{"issue":"8","key":"2026101308064240200_cit16","doi-asserted-by":"publisher","first-page":"1964","DOI":"10.1021\/jasms.0c00425","article-title":"Dissociation of Biomolecules by an\nIntense Low-Energy Electron Beam in a High Sensitivity Time-of-Flight\nMass Spectrometer","volume":"32","author":"Baba","year":"2021","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"6","key":"2026101308064240200_cit17","doi-asserted-by":"publisher","first-page":"750","DOI":"10.1002\/mas.21560","article-title":"Radical solutions:\nPrinciples and application of electron-based dissociation in mass\nspectrometry-based analysis of protein structure","volume":"37","author":"Lermyte","year":"2018","journal-title":"Mass Spectrom. Rev."},{"issue":"14","key":"2026101308064240200_cit18","doi-asserted-by":"publisher","first-page":"8421","DOI":"10.1021\/acs.analchem.8b00984","article-title":"Accurate Sequence Analysis of a Monoclonal\nAntibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying\nMultiple Ion Activation Techniques","volume":"90","author":"Fornelli","year":"2018","journal-title":"Anal. Chem."},{"issue":"2","key":"2026101308064240200_cit19","doi-asserted-by":"publisher","DOI":"10.3390\/antib13020030","article-title":"Enhanced Characterization of Lysine-Linked Antibody\nDrug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy\nCollisional Dissociation-Triggered Electron-Transfer\/Higher-Energy\nCollisional Dissociation and Ultraviolet Photodissociation","volume":"13","author":"Watts","year":"2024","journal-title":"Antibodies."},{"issue":"15","key":"2026101308064240200_cit20","doi-asserted-by":"publisher","first-page":"10246","DOI":"10.1021\/acs.analchem.0c00705","article-title":"Top-Down Characterization of an Intact\nMonoclonal Antibody Using Activated Ion Electron Transfer Dissociation","volume":"92","author":"Lodge","year":"2020","journal-title":"Anal. 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Proteome Res."},{"issue":"9","key":"2026101308064240200_cit22","doi-asserted-by":"publisher","first-page":"5118","DOI":"10.1021\/acs.analchem.4c06350","article-title":"Multilevel\u2013Intact,\nSubunits, and Peptides\u2013Characterization of Antibody-Based Therapeutics\nby a Single-Column LC\u2013MS Setup","volume":"97","author":"Low","year":"2025","journal-title":"Anal.\nChem."},{"issue":"1","key":"2026101308064240200_cit23","doi-asserted-by":"publisher","first-page":"6","DOI":"10.1016\/j.xphs.2019.09.018","article-title":"An Industry Perspective\non Forced Degradation Studies\nof Biopharmaceuticals: Survey Outcome and Recommendations","volume":"109","author":"Halley","year":"2020","journal-title":"J. Pharm. Sci."},{"issue":"29","key":"2026101308064240200_cit24","doi-asserted-by":"publisher","first-page":"8949","DOI":"10.1021\/ja028831n","article-title":"Electron\nCapture Dissociation Initiates\na Free Radical Reaction Cascade","volume":"125","author":"Leymarie","year":"2003","journal-title":"J. Am. Chem.\nSoc."},{"key":"2026101308064240200_cit25","first-page":"1","article-title":"Determination of the NISTmAb Primary\nStructure","volume-title":"State-of-the-Art and Emerging\nTechnologies for Therapeutic Monoclonal Antibody Characterization\nVolume 2. Biopharmaceutical Characterization: The NISTmAb Case Study","author":"Formolo","year":"2015"},{"issue":"10","key":"2026101308064240200_cit26","doi-asserted-by":"publisher","first-page":"2397","DOI":"10.1021\/acs.jproteome.2c00265","article-title":"In Search\nof a Universal Method: A Comparative Survey of Bottom-Up\nProteomics Sample Preparation Methods","volume":"21","author":"Varnavides","year":"2022","journal-title":"J. Proteome\nRes."},{"issue":"5","key":"2026101308064240200_cit27","doi-asserted-by":"publisher","first-page":"1229","DOI":"10.4161\/mabs.29601","article-title":"A novel approach to\ninvestigate the effect of methionine oxidation on pharmacokinetic\nproperties of therapeutic antibodies","volume":"6","author":"Stracke","year":"2014","journal-title":"MAbs"},{"issue":"3","key":"2026101308064240200_cit28","doi-asserted-by":"publisher","first-page":"498","DOI":"10.1080\/19420862.2017.1279773","article-title":"Subunit mass analysis\nfor monitoring antibody oxidation","volume":"9","author":"Sokolowska","year":"2017","journal-title":"mAbs"},{"key":"2026101308064240200_cit29","doi-asserted-by":"publisher","first-page":"1226","DOI":"10.1021\/jasms.6c00016","article-title":"From Congestion to Clarity: On the Complementarity\nof Resolving Power and Spectral Simplification for Intact Protein\nCharacterization","volume":"37","author":"Lieu","year":"2026","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"28","key":"2026101308064240200_cit30","doi-asserted-by":"publisher","first-page":"6463","DOI":"10.1007\/s00216-024-05534-z","article-title":"Towards a universal method for middle-down analysis\nof antibodies via proton transfer charge reduction\u2013Orbitrap\nmass spectrometry","volume":"416","author":"Oates","year":"2024","journal-title":"Anal. Bioanal. Chem."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c03432","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/40\/29262\/68683743\/acs.analchem.6c03432.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/40\/29262\/68683743\/acs.analchem.6c03432.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T12:29:34Z","timestamp":1791894574000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/40\/29262\/5440350\/Middle-Down-Mass-Spectrometry-Characterization-of"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,28]]},"references-count":30,"journal-issue":{"issue":"40","published-online":{"date-parts":[[2026,9,28]]},"published-print":{"date-parts":[[2026,10,13]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c03432","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,28]]}}}