{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T13:07:25Z","timestamp":1788872845410,"version":"build-2803163510"},"reference-count":42,"publisher":"American Chemical Society (ACS)","issue":"35","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Advances in mass spectrometry instrumentation resulted in richer MSn spectra and additional ambiguity in peak assignments. Internal fragment ions (IFs) are often prevalent during top-down mass spectrometry (TDMS) and are particularly difficult to assign\u2500to the extent many practitioners currently forgo their assignment. Accounting for IFs during high-yield dissociation can increase the depth of sequence coverage by an order of magnitude but also results in a geometric increase in the size of the database and ambiguous peak assignments. The ambiguity in TDMS fragment ion assignments comes from the potential for one experimental peak to be assigned to two or more theoretical molecules\u2500most often in the form of isomeric internal fragment ions from distinct sequence regions. The disambiguation of such peaks remains a major challenge in TDMS and is the focus of this work. Ion mobility (IM) mass spectrometry can often separate isomers, including peptides. Starting from a TDMS spectrum containing both unambiguously and ambiguously assigned peaks, we address the hypothesis that ion mobility, or ion mobility followed by fragmentation, can help disambiguate fragment ion assignments by resolving isomeric ambiguity in the mobility domain. We demonstrate that mobility-aligned fragmentation (MAF) can disambiguate TDMS and peptide MS\/MS peak assignments of isomeric terminal and internal ions. Consistent with previous studies, we also demonstrate that a single isomeric species (e.g., an individual b-, a-, or internal ion) can be comprised of multiple conformational isomers with different collisional cross-section (CCS) values.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c03396","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T07:52:34Z","timestamp":1788249154000},"page":"25899-25908","source":"Crossref","is-referenced-by-count":0,"title":["Disambiguation\nof Product Ion Assignments, Including\nInternal Fragment Ions from Top-Down Mass Spectrometry, Using Ion\nMobility-Aligned Fragmentation (MAF)"],"prefix":"10.1021","volume":"98","author":[{"given":"Novera","family":"Alam","sequence":"first","affiliation":[{"name":"Northeastern University , , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Barnett Institute of Chemical and Biological Analysis , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brad J.","family":"Williams","sequence":"additional","affiliation":[{"name":"Waters Corporation , , ,","place":["Milford, Massachusetts, United States, 01757"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Yu","sequence":"additional","affiliation":[{"name":"Northeastern University , , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Barnett Institute of Chemical and Biological Analysis , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Kowalski","sequence":"additional","affiliation":[{"name":"Waters Corporation , , ,","place":["Milford, Massachusetts, United States, 01757"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jane-Marie","family":"Kowalski","sequence":"additional","affiliation":[{"name":"Waters Corporation , , ,","place":["Milford, Massachusetts, United States, 01757"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2645-1873","authenticated-orcid":true,"given":"Jeffrey N.","family":"Agar","sequence":"additional","affiliation":[{"name":"Northeastern University , , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Barnett Institute of Chemical and Biological Analysis , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Northeastern University , , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"issue":"7","key":"2026090808065588800_cit1","doi-asserted-by":"publisher","first-page":"1752","DOI":"10.1021\/jasms.1c00113","article-title":"Internal Fragments\nGenerated from Different Top-Down Mass Spectrometry Fragmentation\nMethods Extend Protein Sequence Coverage","volume":"32","author":"Zenaidee","year":"2021","journal-title":"J.\nAm. 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Chem."},{"issue":"13","key":"2026090808065588800_cit4","doi-asserted-by":"publisher","first-page":"4084","DOI":"10.1039\/C6AN00453A","article-title":"Modular calibrant\nsets for the structural analysis of nucleic acids by ion mobility\nspectrometry mass spectrometry","volume":"141","author":"Lippens","year":"2016","journal-title":"Analyst"},{"issue":"10","key":"2026090808065588800_cit5","doi-asserted-by":"publisher","first-page":"3335","DOI":"10.1039\/C5AN00152H","article-title":"Non-derivatized\nglycan analysis by\nreverse phase liquid chromatography and ion mobility-mass spectrometry","volume":"140","author":"Lareau","year":"2015","journal-title":"Analyst"},{"issue":"10","key":"2026090808065588800_cit6","doi-asserted-by":"publisher","first-page":"5138","DOI":"10.1021\/ac400403d","article-title":"Ion mobility-mass\nspectrometry of complex carbohydrates:\ncollision cross sections of sodiated N-linked glycans","volume":"85","author":"Pagel","year":"2013","journal-title":"Anal. Chem."},{"issue":"7572","key":"2026090808065588800_cit7","doi-asserted-by":"publisher","first-page":"241","DOI":"10.1038\/nature15388","article-title":"Identification of carbohydrate\nanomers using ion mobility-mass spectrometry","volume":"526","author":"Hofmann","year":"2015","journal-title":"Nature"},{"issue":"5","key":"2026090808065588800_cit8","doi-asserted-by":"publisher","first-page":"1649","DOI":"10.1039\/C5AN02062J","article-title":"Uncovering biologically\nsignificant lipid isomers with liquid chromatography, ion mobility\nspectrometry and mass spectrometry","volume":"141","author":"Kyle","year":"2016","journal-title":"Analyst"},{"issue":"1","key":"2026090808065588800_cit9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.ijms.2006.07.021","article-title":"An investigation\nof the mobility separation of some peptide and protein ions using\na new hybrid quadrupole\/travelling wave IMS\/oa-ToF instrument","volume":"261","author":"Pringle","year":"2007","journal-title":"Int. J. Mass Spectrom."},{"issue":"10","key":"2026090808065588800_cit10","doi-asserted-by":"publisher","first-page":"2396","DOI":"10.1002\/slct.201600460","article-title":"Ion Mobility Separations\nof Isomers based upon Long Path Length Structures for Lossless Ion\nManipulations Combined with Mass Spectrometry","volume":"1","author":"Deng","year":"2016","journal-title":"ChemistrySelect"},{"key":"2026090808065588800_cit11","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/978-1-0716-0030-6_1","article-title":"Fundamentals of\nIon Mobility-Mass Spectrometry for the Analysis of Biomolecules","volume":"2084","author":"Morris","year":"2020","journal-title":"Methods Mol. 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Mass\nSpectrom."},{"issue":"16","key":"2026090808065588800_cit13","doi-asserted-by":"publisher","first-page":"2766","DOI":"10.1002\/pmic.201500048","article-title":"Enhancing bottom-up\nand top-down\nproteomic measurements with ion mobility separations","volume":"15","author":"Baker","year":"2015","journal-title":"Proteomics"},{"issue":"4","key":"2026090808065588800_cit14","doi-asserted-by":"publisher","first-page":"2107","DOI":"10.1021\/ac4038448","article-title":"Conformational ordering of biomolecules\nin the gas phase: nitrogen collision cross sections measured on a\nprototype high resolution drift tube ion mobility-mass spectrometer","volume":"86","author":"May","year":"2014","journal-title":"Anal. Chem."},{"issue":"1","key":"2026090808065588800_cit15","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1039\/C0AN00373E","article-title":"How useful is ion\nmobility mass spectrometry for structural\nbiology? The relationship between protein crystal structures and their\ncollision cross sections in the gas phase","volume":"136","author":"Jurneczko","year":"2011","journal-title":"Analyst"},{"issue":"11","key":"2026090808065588800_cit16","doi-asserted-by":"publisher","first-page":"2185","DOI":"10.1007\/s13361-019-02288-2","article-title":"Ion Mobility Spectrometry:\nFundamental Concepts, Instrumentation,\nApplications, and the Road Ahead","volume":"30","author":"Dodds","year":"2019","journal-title":"J. Am. Soc.\nMass Spectrom."},{"issue":"11","key":"2026090808065588800_cit17","doi-asserted-by":"publisher","first-page":"1559","DOI":"10.1002\/rcm.5013","article-title":"Enhancements\nin travelling wave ion\nmobility resolution","volume":"25","author":"Giles","year":"2011","journal-title":"Rapid Commun. 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Exp."},{"issue":"7","key":"2026090808065588800_cit28","doi-asserted-by":"publisher","first-page":"1010","DOI":"10.1039\/C7AN00031F","article-title":"New frontiers\nfor\nmass spectrometry based upon structures for lossless ion manipulations","volume":"142","author":"Ibrahim","year":"2017","journal-title":"Analyst"},{"key":"2026090808065588800_cit29","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1016\/j.cbpa.2017.10.022","article-title":"Fundamentals of ion\nmobility spectrometry","volume":"42","author":"Gabelica","year":"2018","journal-title":"Curr. Opin. Chem. Biol."},{"key":"2026090808065588800_cit30","doi-asserted-by":"publisher","first-page":"100138","DOI":"10.1016\/j.mcpro.2021.100138","article-title":"Trapped Ion\nMobility Spectrometry\nand Parallel Accumulation-Serial Fragmentation in Proteomics","volume":"20","author":"Meier","year":"2021","journal-title":"Mol. Cell. Proteomics"},{"issue":"9","key":"2026090808065588800_cit31","doi-asserted-by":"publisher","first-page":"3888","DOI":"10.1021\/acs.analchem.1c04870","article-title":"Enhanced Top-Down\nProtein Characterization with Electron Capture Dissociation and Cyclic\nIon Mobility Spectrometry","volume":"94","author":"Shaw","year":"2022","journal-title":"Anal. Chem."},{"issue":"7","key":"2026090808065588800_cit32","doi-asserted-by":"publisher","first-page":"1204","DOI":"10.1021\/jasms.2c00053","article-title":"Toward Rapid Aspartic Acid Isomer Localization in Therapeutic Peptides\nUsing Cyclic Ion Mobility Mass Spectrometry","volume":"33","author":"Gibson","year":"2022","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"6","key":"2026090808065588800_cit33","doi-asserted-by":"publisher","first-page":"477","DOI":"10.1016\/1044-0305(93)80005-J","article-title":"Dehydration of peptide\n[M + H]+ ions in the gas phase","volume":"4","author":"Ballard","year":"1993","journal-title":"J. Am. Soc.\nMass Spectrom."},{"issue":"52","key":"2026090808065588800_cit34","doi-asserted-by":"publisher","first-page":"17774","DOI":"10.1021\/ja805074d","article-title":"Sequence-Scrambling\nFragmentation\nPathways of Protonated Peptides","volume":"130","author":"Bleiholder","year":"2008","journal-title":"J. Am. Chem.\nSoc."},{"issue":"6","key":"2026090808065588800_cit35","doi-asserted-by":"publisher","first-page":"1286","DOI":"10.1021\/jp0763937","article-title":"On the\ndynamics of fragment isomerization in collision-induced dissociation\nof peptides","volume":"112","author":"Polfer","year":"2008","journal-title":"J. Phys. Chem. A"},{"issue":"23","key":"2026090808065588800_cit36","doi-asserted-by":"publisher","first-page":"8146","DOI":"10.1021\/acs.analchem.1c05171","article-title":"Tandem Trapped\nIon Mobility Spectrometry\/Mass Spectrometry\n(tTIMS\/MS) Reveals Sequence-Specific Determinants of Top-Down Protein\nFragment Ion Cross Sections","volume":"94","author":"Liu","year":"2022","journal-title":"Anal. Chem."},{"key":"2026090808065588800_cit37","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1016\/j.ijms.2015.07.021","article-title":"Proposed nomenclature for peptide\nion fragmentation","volume":"390","author":"Chu","year":"2015","journal-title":"Int. J. Mass Spectrom."},{"issue":"24","key":"2026090808065588800_cit38","doi-asserted-by":"publisher","first-page":"9540","DOI":"10.1021\/ac202327r","article-title":"Statistical analysis\nof electron transfer dissociation\npairwise fragmentation patterns","volume":"83","author":"Li","year":"2011","journal-title":"Anal. Chem."},{"issue":"7","key":"2026090808065588800_cit39","doi-asserted-by":"publisher","first-page":"1291","DOI":"10.1016\/j.jasms.2007.03.029","article-title":"Backbone cleavages and sequential\nloss of carbon monoxide and ammonia from protonated AGG: a combined\ntandem mass spectrometry, isotope labeling, and theoretical study","volume":"18","author":"Bythell","year":"2007","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"32","key":"2026090808065588800_cit40","doi-asserted-by":"publisher","first-page":"10364","DOI":"10.1021\/ja062440h","article-title":"Scrambling\nof Sequence Information in Collision-Induced Dissociation of Peptides","volume":"128","author":"Harrison","year":"2006","journal-title":"J. Am. Chem. Soc."},{"issue":"7","key":"2026090808065588800_cit41","doi-asserted-by":"publisher","first-page":"2669","DOI":"10.1021\/pr070121z","article-title":"Relative specificities\nof water and ammonia losses from backbone fragments in collision-activated\ndissociation","volume":"6","author":"Savitski","year":"2007","journal-title":"J. Proteome Res."},{"issue":"16","key":"2026090808065588800_cit42","doi-asserted-by":"publisher","DOI":"10.1002\/rcm.70104","article-title":"Combinatorial\nProbabilities of Multiple\nFragmentation Events Explain Polypeptide MS\/MS Intensity Distribution,\nOverrepresentation of Smaller Fragments, and Missing Middle of Top-Down\nMS","volume":"40","author":"Yang","year":"2026","journal-title":"Rapid Commun. Mass Spectrom."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c03396","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/35\/25899\/67412919\/acs.analchem.6c03396.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/35\/25899\/67412919\/acs.analchem.6c03396.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T12:13:18Z","timestamp":1788869598000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/35\/25899\/5329326\/Disambiguation-of-Product-Ion-Assignments"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,26]]},"references-count":42,"journal-issue":{"issue":"35","published-online":{"date-parts":[[2026,8,26]]},"published-print":{"date-parts":[[2026,9,8]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c03396","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,26]]}}}