{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T15:10:16Z","timestamp":1790694616202,"version":"4.1.0"},"reference-count":39,"publisher":"American Chemical Society (ACS)","issue":"1","license":[{"start":{"date-parts":[[2020,10,13]],"date-time":"2020-10-13T00:00:00Z","timestamp":1602547200000},"content-version":"vor","delay-in-days":28,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100010661","name":"European Commission","doi-asserted-by":"publisher","award":["EPIC-XS project number 823839"],"award-info":[{"award-number":["EPIC-XS project number 823839"]}],"id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001665","name":"Agence Nationale de la Recherche","doi-asserted-by":"publisher","award":["ANR-15-CE18-0021"],"award-info":[{"award-number":["ANR-15-CE18-0021"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003762","name":"Institut Pasteur","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/501100003762","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004794","name":"Centre National de la Recherche Scientifique","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/501100004794","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"PasteurInnov","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,1,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The current technique used for microbial identification in hospitals is matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, it suffers from important limitations, in particular, for closely related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a liquid chromatography (LC)\u2013MS\/MS top-down proteomics platform, which aims at discriminating closely related pathogenic bacteria through the identification of specific proteoforms. Using Escherichia coli as a model, all steps of the workflow were optimized: protein extraction, on-line LC separation, MS method, and data analysis. Using optimized parameters, about 220 proteins, corresponding to more than 500 proteoforms, could be identified in a single run. We then used this platform for the discrimination of enterobacterial pathogens undistinguishable by MALDI-TOF, although leading to very different clinical outcomes. For each pathogen, we identified specific proteoforms that could potentially be used as biomarkers. We also improved the characterization of poorly described bacterial strains. Our results highlight the advantage of addressing proteoforms rather than peptides for accurate bacterial characterization and qualify top-down proteomics as a promising tool in clinical microbiology. Data are available via ProteomeXchange with the identifier PXD019247.<\/jats:p>","DOI":"10.1021\/acs.jproteome.0c00351","type":"journal-article","created":{"date-parts":[[2020,9,15]],"date-time":"2020-09-15T06:49:47Z","timestamp":1600152587000},"page":"202-211","source":"Crossref","is-referenced-by-count":50,"title":["Optimization of a Top-Down Proteomics Platform for\nClosely Related Pathogenic Bacterial Discrimination"],"prefix":"10.1021","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1845-0048","authenticated-orcid":true,"given":"Mathieu","family":"Dupre\u0301","sequence":"first","affiliation":[{"name":"Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magalie","family":"Duchateau","sequence":"additional","affiliation":[{"name":"Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Malosse","sequence":"additional","affiliation":[{"name":"Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diogo","family":"Borges-Lima","sequence":"additional","affiliation":[{"name":"Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria","family":"Calvaresi","sequence":"additional","affiliation":[{"name":"Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabelle","family":"Podglajen","sequence":"additional","affiliation":[{"name":"Assistance Publique-Ho\u0302pitaux de Paris , , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominique","family":"Clermont","sequence":"additional","affiliation":[{"name":"Collection of the Institut Pasteur (CIP), Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martial","family":"Rey","sequence":"additional","affiliation":[{"name":"Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9427-543X","authenticated-orcid":true,"given":"Julia","family":"Chamot-Rooke","sequence":"additional","affiliation":[{"name":"Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur , ,","place":["Paris, France, 75015"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,9,15]]},"reference":[{"key":"2026090223354975100_cit1","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1146\/annurev.anchem.1.031207.112838","article-title":"Mass spectrometry for\nrapid characterization of microorganisms","volume":"1","author":"Demirev","year":"2008","journal-title":"Annu.\nRev. Anal. Chem."},{"key":"2026090223354975100_cit2","doi-asserted-by":"publisher","first-page":"552","DOI":"10.1086\/600886","article-title":"A revolution\nin the identification of pathogens in clinical laboratories","volume":"49","author":"Nassif","year":"2009","journal-title":"Clin. Infect. Dis."},{"key":"2026090223354975100_cit3","doi-asserted-by":"publisher","first-page":"791","DOI":"10.3389\/fmicb.2015.00791","article-title":"MALDI-TOF mass spectrometry:\nan emerging technology for microbial identification and diagnosis","volume":"6","author":"Singhal","year":"2015","journal-title":"Front. Microbiol."},{"key":"2026090223354975100_cit4","doi-asserted-by":"publisher","first-page":"447","DOI":"10.1016\/j.tim.2016.12.006","article-title":"Can MALDI-TOF Mass\nSpectrometry Reasonably Type Bacteria?","volume":"25","author":"Sauget","year":"2017","journal-title":"Trends\nMicrobiol."},{"key":"2026090223354975100_cit5","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1016\/j.mimet.2014.02.015","article-title":"Insufficient\ndiscriminatory power\nof MALDI-TOF mass spectrometry for typing of Enterococcus faecium\nand Staphylococcus aureus isolates","volume":"100","author":"Lasch","year":"2014","journal-title":"J. Microbiol.\nMethods"},{"key":"2026090223354975100_cit6","doi-asserted-by":"publisher","first-page":"13944","DOI":"10.1038\/srep13944","article-title":"Rapid Bacterial\nIdentification, Resistance, Virulence and Type Profiling using Selected\nReaction Monitoring Mass Spectrometry","volume":"5","author":"Charretier","year":"2015","journal-title":"Sci.\nRep."},{"key":"2026090223354975100_cit7","doi-asserted-by":"publisher","first-page":"1985","DOI":"10.3389\/fmicb.2019.01985","article-title":"Evaluation of Sample Preparation Methods for Fast Proteotyping\nof Microorganisms by Tandem Mass Spectrometry","volume":"10","author":"Hayoun","year":"2019","journal-title":"Front. Microbiol."},{"key":"2026090223354975100_cit8","doi-asserted-by":"publisher","first-page":"1419","DOI":"10.1074\/mcp.r500012-mcp200","article-title":"Interpretation\nof Shotgun Proteomic Data","volume":"4","author":"Nesvizhskii","year":"2005","journal-title":"Mol. Cell. 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Proteomics"},{"key":"2026090223354975100_cit28","doi-asserted-by":"publisher","first-page":"796","DOI":"10.1074\/mcp.ra118.000993","article-title":"Accurate\nEstimation of Context-Dependent\nFalse Discovery Rates in Top-Down Proteomics","volume":"18","author":"LeDuc","year":"2019","journal-title":"Mol. Cell. Proteomics"},{"key":"2026090223354975100_cit29","doi-asserted-by":"publisher","first-page":"4152","DOI":"10.1021\/acs.analchem.5b00162","article-title":"Benchmarking\nmultiple fragmentation methods on an orbitrap\nfusion for top-down phospho-proteoform characterization","volume":"87","author":"Brunner","year":"2015","journal-title":"Anal. Chem."},{"key":"2026090223354975100_cit30","doi-asserted-by":"publisher","first-page":"4308","DOI":"10.1021\/pr3004216","article-title":"Evaluation of the\ncompact high-field orbitrap for top-down\nproteomics of human cells","volume":"11","author":"Ahlf","year":"2012","journal-title":"J. Proteome Res."},{"key":"2026090223354975100_cit31","doi-asserted-by":"publisher","first-page":"465","DOI":"10.1007\/978-1-4939-0685-7_31","article-title":"Top-down proteomics\nby means of Orbitrap mass spectrometry","volume":"1156","author":"Scheffler","year":"2014","journal-title":"Methods\nMol. Biol."},{"key":"2026090223354975100_cit32","doi-asserted-by":"publisher","first-page":"1138","DOI":"10.1021\/acs.jproteome.7b00801","article-title":"Top-Down Characterization\nof Heavily Modified Histones\nUsing 193 nm Ultraviolet Photodissociation Mass Spectrometry","volume":"17","author":"Greer","year":"2018","journal-title":"J. Proteome Res."},{"key":"2026090223354975100_cit33","doi-asserted-by":"publisher","first-page":"12519","DOI":"10.1021\/acs.analchem.8b02344","article-title":"Maximizing Sequence Coverage in Top-Down\nProteomics By Automated Multimodal Gas-Phase Protein Fragmentation","volume":"90","author":"Shliaha","year":"2018","journal-title":"Anal. 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