{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T15:03:55Z","timestamp":1785942235242,"version":"3.56.0"},"reference-count":45,"publisher":"American Chemical Society (ACS)","issue":"8","license":[{"start":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T00:00:00Z","timestamp":1784246400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000266","name":"Engineering and Physical Sciences Research Council","doi-asserted-by":"publisher","award":["EP\/Y004604\/1"],"award-info":[{"award-number":["EP\/Y004604\/1"]}],"id":[{"id":"10.13039\/501100000266","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["305669\/Z\/23\/Z"],"award-info":[{"award-number":["305669\/Z\/23\/Z"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Here, we demonstrate PTCR-HCD MS3 as a tool for improving protein identification in native ambient mass spectrometry (NAMS). NAMS involves the direct analysis of tissue sections with no or little sample preparation and no (solution-phase) separation postsampling. NAMS samples are therefore highly complex, comprising proteins, salts, and other biomolecules, and NAMS spectra are highly congested. PTCR MS has emerged as a useful strategy in top-down proteomics. Most commonly, it is applied as an adjunct MS2 step, providing molecular weight information to support sequence information provided by collision-based MS2 or it is applied as an MS3 step to simplify collision, photon, or electron-based MS2 by reducing m\/z overlap of product ions. In this work, we show that the selection of charge-reduced PTCR product ions for subsequent HCD reduces the incidence of chimeric fragmentation spectra and improves confidence in protein identification in NAMS samples from mouse spinal cord, rat kidney, and cell-line-derived xenograft tissue. As well as removing interference from overlapping protein species, we show that PTCR-HCD MS3 also removes interference from overlapping lipid species which can similarly affect confident protein identification. The approach also allows identification of proteins that are masked at the MS1 level but are revealed following PTCR.<\/jats:p>","DOI":"10.1021\/jasms.6c00165","type":"journal-article","created":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T17:39:21Z","timestamp":1784309961000},"page":"1971-1980","source":"Crossref","is-referenced-by-count":0,"title":["Improved Protein Identification\nin Native Ambient\nMass Spectrometry by the Integration of Proton Transfer Charge Reduction\nand Higher-Energy Collision Dissociation (PTCR-HCD MS3)"],"prefix":"10.1021","volume":"37","author":[{"given":"Rebecca L.","family":"Edwards","sequence":"first","affiliation":[{"name":"University of Birmingham , , Edgbaston , ,","place":["Birmingham, U.K., B15 2TT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2286-5780","authenticated-orcid":true,"given":"Oliver J.","family":"Hale","sequence":"additional","affiliation":[{"name":"University of Birmingham , , Edgbaston , ,","place":["Birmingham, U.K., B15 2TT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-0157-9181","authenticated-orcid":true,"given":"Sarah R. W.","family":"Vickers","sequence":"additional","affiliation":[{"name":"University of Birmingham , , Edgbaston , ,","place":["Birmingham, U.K., B15 2TT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4590-9384","authenticated-orcid":true,"given":"Helen J.","family":"Cooper","sequence":"additional","affiliation":[{"name":"University of Birmingham , , Edgbaston , ,","place":["Birmingham, U.K., B15 2TT"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,17]]},"reference":[{"issue":"22","key":"2026080510112120400_cit1","doi-asserted-by":"publisher","first-page":"8534","DOI":"10.1021\/ja00022a058","article-title":"Observation of the\nHeme-Globin Complex in Native Myoglobin\nby Electrospray-Ionization Mass Spectrometry","volume":"113","author":"Katta","year":"1991","journal-title":"J. Am. Chem. Soc."},{"issue":"20","key":"2026080510112120400_cit2","doi-asserted-by":"publisher","first-page":"7818","DOI":"10.1021\/ja00020a085","article-title":"Observation of Noncovalent Enzyme-Substrate\nand Enyzme-Product Complexes by Ion-Spray Spectrometry","volume":"113","author":"Ganem","year":"1991","journal-title":"J. Am. Chem. Soc."},{"issue":"8","key":"2026080510112120400_cit3","doi-asserted-by":"publisher","first-page":"7269","DOI":"10.1021\/acs.chemrev.1c00212","article-title":"High-Resolution Native Mass Spectrometry","volume":"122","author":"Tamara","year":"2022","journal-title":"Chem. Rev."},{"issue":"5","key":"2026080510112120400_cit4","doi-asserted-by":"publisher","first-page":"505","DOI":"10.1038\/s41592-020-0821-0","article-title":"Combining native and \u2019omics\u2019\nmass spectrometry to identify endogenous ligands bound to membrane\nproteins","volume":"17","author":"Gault","year":"2020","journal-title":"Nat. Methods"},{"issue":"12","key":"2026080510112120400_cit5","doi-asserted-by":"publisher","first-page":"2388","DOI":"10.1038\/s41592-024-02279-6","article-title":"Top-down mass spectrometry\nof native\nproteoforms and their complexes: a community study","volume":"21","author":"Habeck","year":"2024","journal-title":"Nat. Methods"},{"issue":"5695","key":"2026080510112120400_cit6","doi-asserted-by":"publisher","first-page":"471","DOI":"10.1126\/science.1104404","article-title":"Mass spectrometry\nsampling under ambient conditions with desorption electrospray ionization","volume":"306","author":"Takats","year":"2004","journal-title":"Science"},{"issue":"4","key":"2026080510112120400_cit7","doi-asserted-by":"publisher","first-page":"437","DOI":"10.1002\/mas.20346","article-title":"Characterization\nof proteins by ambient mass spectrometry","volume":"31","author":"Yao","year":"2012","journal-title":"Mass\nSpectrom Rev."},{"issue":"8","key":"2026080510112120400_cit8","doi-asserted-by":"publisher","first-page":"1320","DOI":"10.1007\/s13361-015-1152-8","article-title":"Native Liquid Extraction\nSurface Analysis Mass Spectrometry: Analysis of Noncovalent Protein\nComplexes Directly from Dried Substrates","volume":"26","author":"Martin","year":"2015","journal-title":"J.\nAm. Soc. Mass Spectrom."},{"issue":"12","key":"2026080510112120400_cit9","doi-asserted-by":"publisher","first-page":"2531","DOI":"10.1021\/jasms.0c00226","article-title":"Native Mass Spectrometry\nImaging and In Situ Top-Down\nIdentification of Intact Proteins Directly from Tissue","volume":"31","author":"Hale","year":"2020","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"5","key":"2026080510112120400_cit10","doi-asserted-by":"publisher","first-page":"2120","DOI":"10.1021\/jacs.1c10032","article-title":"Native Ambient Mass\nSpectrometry Imaging of Ligand-Bound and Metal-Bound Proteins in Rat\nBrain","volume":"144","author":"Sisley","year":"2022","journal-title":"J. Am. Chem. Soc."},{"issue":"10","key":"2026080510112120400_cit11","doi-asserted-by":"publisher","first-page":"4619","DOI":"10.1021\/acs.analchem.0c05277","article-title":"Native Mass Spectrometry\nImaging of Proteins and Protein\nComplexes by Nano-DESI","volume":"93","author":"Hale","year":"2021","journal-title":"Anal. Chem."},{"issue":"11","key":"2026080510112120400_cit12","doi-asserted-by":"publisher","first-page":"6962","DOI":"10.1021\/acs.analchem.9b00971","article-title":"Direct Mass Spectrometry\nAnalysis of Protein Complexes and Intact Proteins up to &gt; 70 kDa\nfrom\nTissue","volume":"91","author":"Griffiths","year":"2019","journal-title":"Anal. Chem."},{"issue":"14","key":"2026080510112120400_cit13","doi-asserted-by":"publisher","first-page":"5608","DOI":"10.1021\/acs.analchem.1c05353","article-title":"Native Ambient Mass\nSpectrometry Enables Analysis of Intact Endogenous Protein Assemblies\nup to 145 kDa Directly from Tissue","volume":"94","author":"Hale","year":"2022","journal-title":"Anal. Chem."},{"issue":"15","key":"2026080510112120400_cit14","doi-asserted-by":"publisher","first-page":"5723","DOI":"10.1039\/D3SC04933G","article-title":"Laser capture microdissection\nand native mass spectrometry for spatially-resolved analysis of intact\nprotein assemblies in tissue","volume":"15","author":"Hughes","year":"2024","journal-title":"Chem. Sci."},{"issue":"29","key":"2026080510112120400_cit15","doi-asserted-by":"publisher","first-page":"15658","DOI":"10.1021\/jacs.3c03454","article-title":"Tissue Washing Improves\nNative Ambient Mass Spectrometry Detection of Membrane Proteins Directly\nfrom Tissue","volume":"145","author":"Sisley","year":"2023","journal-title":"J. Am. Chem. Soc."},{"key":"2026080510112120400_cit16","doi-asserted-by":"publisher","first-page":"28343","DOI":"10.1021\/acs.analchem.5c05767","article-title":"Imaging of protein assemblies up to 231 kDa in tissues\nwith nano-DESI mass spectrometry","volume":"97","author":"Hale","year":"2025","journal-title":"Anal. Chem."},{"issue":"3","key":"2026080510112120400_cit17","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1038\/nmeth.2369","article-title":"Consortium for Top\nDown, P., Proteoform: a single term\ndescribing protein complexity","volume":"10","author":"Smith","year":"2013","journal-title":"Nat. Methods"},{"key":"2026080510112120400_cit18","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.ijms.2017.10.009","article-title":"Native\nmass spectrometry imaging of intact proteins\nand protein complexes in thin tissue sections","volume":"437","author":"Griffiths","year":"2019","journal-title":"Int. J. Mass Spectrom."},{"key":"2026080510112120400_cit19","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-50514-7","article-title":"Mass spectrometry\nimaging of SOD1 protein-metal complexes in SOD1G93A transgenic mice\nimplicates demetalation with pathology","volume":"15","author":"Hale","year":"2024","journal-title":"Nat.\nCommun."},{"issue":"48","key":"2026080510112120400_cit20","doi-asserted-by":"publisher","first-page":"12918","DOI":"10.1039\/D0SC04392C","article-title":"Higher-order structural\ncharacterisation\nof native proteins and complexes by top-down mass spectrometry","volume":"11","author":"Zhou","year":"2020","journal-title":"Chem. Sci."},{"issue":"6","key":"2026080510112120400_cit21","doi-asserted-by":"publisher","first-page":"7778","DOI":"10.1093\/bioinformatics\/btad359","article-title":"MASH Native: a unified\nsolution for native top-down proteomics data processing","volume":"39","author":"Larson","year":"2023","journal-title":"Bioinformatics"},{"issue":"4","key":"2026080510112120400_cit22","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbapap.2022.140758","article-title":"Characterization\nof large intact protein ions by mass\nspectrometry: What directions should we follow?","volume":"1870","author":"Fornelli","year":"2022","journal-title":"Biochim. Biophys. Acta Proteins Proteom."},{"issue":"1","key":"2026080510112120400_cit23","doi-asserted-by":"publisher","first-page":"252","DOI":"10.1021\/acs.analchem.9b05014","article-title":"Recent Developments\nin Gas-Phase Ion\/Ion Reactions\nfor Analytical Mass Spectrometry","volume":"92","author":"Foreman","year":"2020","journal-title":"Anal. Chem."},{"issue":"8","key":"2026080510112120400_cit24","doi-asserted-by":"publisher","first-page":"1548","DOI":"10.1021\/acscentsci.4c00462","article-title":"To 200,000 m\/z and Beyond: Native\nElectron Capture Charge Reduction Mass Spectrometry Deconvolves Heterogeneous\nSignals in Large Biopharmaceutical Analytes","volume":"10","author":"Le Huray","year":"2024","journal-title":"ACS Cent. Sci."},{"key":"2026080510112120400_cit25","doi-asserted-by":"publisher","first-page":"1537","DOI":"10.1021\/acscentsci.4c00461","article-title":"Protein complex\nheterogeneity and topology revealed by electron capture charge reduction\nand surface induced dissociation","volume":"10","author":"Shaw","year":"2024","journal-title":"ACS Cent.\nSci."},{"issue":"27","key":"2026080510112120400_cit26","doi-asserted-by":"publisher","first-page":"9463","DOI":"10.1073\/pnas.0503189102","article-title":"Protein identification using sequential\nion\/ion reactions and tandem mass spectrometry","volume":"102","author":"Coon","year":"2005","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"22","key":"2026080510112120400_cit27","doi-asserted-by":"publisher","first-page":"4026","DOI":"10.1021\/ac9605657","article-title":"Ion\/ion\nproton transfer reactions\nfor protein mixture analysis","volume":"68","author":"Stephenson","year":"1996","journal-title":"Anal. Chem."},{"issue":"1","key":"2026080510112120400_cit28","doi-asserted-by":"publisher","first-page":"1041","DOI":"10.1021\/acs.analchem.9b04026","article-title":"Enhanced Sequence Coverage of Large\nProteins by Combining Ultraviolet Photodissociation with Proton Transfer\nReactions","volume":"92","author":"Sanders","year":"2020","journal-title":"Anal. Chem."},{"issue":"24","key":"2026080510112120400_cit29","doi-asserted-by":"publisher","first-page":"15732","DOI":"10.1021\/acs.analchem.9b03925","article-title":"Proton Transfer\nCharge Reduction Enables High-Throughput Top-Down Analysis of Large\nProteoforms","volume":"91","author":"Huguet","year":"2019","journal-title":"Anal. Chem."},{"key":"2026080510112120400_cit30","doi-asserted-by":"publisher","first-page":"2334","DOI":"10.1021\/jasms.1c00062","article-title":"Sequential\nion-ion reactions for\nenhanced gas-phase sequencing of large intact proteins in a tribrid\nmass spectrometer","volume":"32","author":"Kline","year":"2021","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"2026080510112120400_cit31","doi-asserted-by":"publisher","first-page":"1289","DOI":"10.1021\/jasms.6c00060","article-title":"Proton transfer charge\nreduction enables isobaric labeling-based\nproteoform quantification of overlapping signals in top-down mass\nspectrometry","volume":"37","author":"Kaulich","year":"2026","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"2026080510112120400_cit32","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1016\/j.ijms.2006.09.030","article-title":"Analysis of intact proteins on a\nchromatographic time\nscale by electron transfer dissociation tandem mass spectrometry","volume":"259","author":"Chi","year":"2007","journal-title":"Int. J. Mass Spectrom."},{"key":"2026080510112120400_cit33","doi-asserted-by":"publisher","first-page":"617","DOI":"10.1016\/j.ijms.2014.06.023","article-title":"Protein derivatization and sequential\nion\/ion reacions\nto enhance sequence coverage produced by electron transfer dissociation\nmass spectrometry","volume":"377","author":"Anderson","year":"2015","journal-title":"Int. J. Mass Spectrom."},{"key":"2026080510112120400_cit34","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202201458","article-title":"Native ambient mass\nspectrometry of an intact membrane\nprotein assembly and soluble protein assemblies directly from lens\ntissue","volume":"61","author":"Hale","year":"2022","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026080510112120400_cit35","doi-asserted-by":"publisher","first-page":"2163","DOI":"10.1007\/s13361-019-02290-8","article-title":"Ion-ion proton transfer\nand parallel\nion parking for the analysis of mixtures of intact proteins on a modified\norbitra mass analyzer","volume":"30","author":"Ugrin","year":"2019","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"2026080510112120400_cit36","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0023244","article-title":"Optimised\nand rapid\npre-clinial screening in the SOD1G93A transgenic mouse\nmodel of amyotrophic lateral sclerosis (ALS)","volume":"6","author":"Mead","year":"2011","journal-title":"PLoS One"},{"key":"2026080510112120400_cit37","doi-asserted-by":"publisher","first-page":"15658","DOI":"10.1021\/jacs.3c03454","article-title":"Tissue washing improves\nnative ambient mass spectrometry detection of membrane proteins directly\nfrom tissue","volume":"145","author":"Sisley","year":"2023","journal-title":"J. Am. Chem. Soc."},{"key":"2026080510112120400_cit38","doi-asserted-by":"publisher","first-page":"763","DOI":"10.1021\/jasms.9b00037","article-title":"Voltage\nrollercoaster filtering of low-mass contaminants during native protein\nanalysis","volume":"31","author":"McGee","year":"2020","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"2026080510112120400_cit39","doi-asserted-by":"publisher","first-page":"4370","DOI":"10.1021\/acs.analchem.5b00140","article-title":"Bayesian deconvolution\nof mass and ion mobility spectra:\nFrom inary interactions to polydisperse ensembles","volume":"87","author":"Marty","year":"2015","journal-title":"Anal. Chem."},{"key":"2026080510112120400_cit40","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1109\/MCSE.2007.55","article-title":"Matplotlib:\nA 2D graphics environment","volume":"9","author":"Hunter","year":"2007","journal-title":"Comput. Sci. Eng."},{"key":"2026080510112120400_cit41","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-50514-7","article-title":"Mass spectrometry\nimaging of SOD1 protein-metal complexes in SOD1G93A transgenic mice\nimplicates demetalation with pathology","volume":"15","author":"Hale","year":"2024","journal-title":"Nat.\nCommun."},{"key":"2026080510112120400_cit42","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-47693-8","article-title":"Exposing the\nmolecular heterogeneity of glycosylated biotherapeutics","volume":"15","author":"Schachner","year":"2024","journal-title":"Nat. Commun."},{"key":"2026080510112120400_cit43","doi-asserted-by":"publisher","first-page":"1203","DOI":"10.1007\/s13361-017-1635-x","article-title":"Defining\ngas-phase fragmentation propensities of intact proteins during native\ntop-down mass spectrometry","volume":"28","author":"Haverland","year":"2017","journal-title":"J. Am. Soc. Mass\nSpectrom."},{"key":"2026080510112120400_cit44","doi-asserted-by":"publisher","first-page":"1398","DOI":"10.1021\/jasms.0c00026","article-title":"Using 10,000 fragment\nions to inform\nscoring in native top-down proteomics","volume":"31","author":"Ives","year":"2020","journal-title":"J. Am.\nSoc. Mass Spectrom."},{"key":"2026080510112120400_cit45","doi-asserted-by":"publisher","first-page":"7150","DOI":"10.1021\/ac502040v","article-title":"MultiNotch\nMS3 enables accurate, sensitive, and multiplexed detection of differential\nexpression across cancer cell line proteomes","volume":"86","author":"McAlister","year":"2014","journal-title":"Anal. Chem."}],"container-title":["Journal of\nthe American\nSociety for Mass Spectrometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jasms.6c00165","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/8\/1971\/66014129\/jasms.6c00165.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/8\/1971\/66014129\/jasms.6c00165.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T14:25:54Z","timestamp":1785939954000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jamsef\/article\/37\/8\/1971\/5214721\/Improved-Protein-Identification-in-Native-Ambient"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,17]]},"references-count":45,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,7,17]]},"published-print":{"date-parts":[[2026,8,5]]}},"URL":"https:\/\/doi.org\/10.1021\/jasms.6c00165","relation":{},"ISSN":["1044-0305","1879-1123"],"issn-type":[{"value":"1044-0305","type":"print"},{"value":"1879-1123","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,17]]}}}