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Matrix\u2010assisted laser desorption\/ionization time\u2010of\u2010flight mass spectrometry (MALDI\u2010TOF MS) of intact cells enables fast identification of the microorganisms based on their mass spectrometry protein fingerprint profiles. However, the MALDI\u2010TOF MS examination must be preceded by a time\u2010demanding cultivation of the native bacteria to isolate representative cell samples to obtain indicative fingerprints. Isoelectric focusing (IEF) is capable of separating bacterial cells according to their isoelectric point while effectively removing other non\u2010focusing compounds from sample matrix. In this work, we present a divergent\u2010flow IEF chip (DF\u2010IEF chip) fractionation as an alternative way for sample clean\u2010up and concentration of bacterial cells to prepare samples usable for following MALDI\u2010TOF MS analysis without the need of time\u2010demanding cultivation. By means of DF\u2010IEF chip method, we processed four species of highly pathogenic bacteria (<jats:italic>Bacillus anthracis<\/jats:italic>, <jats:italic>Brucella abortus<\/jats:italic>, <jats:italic>Burkholderia mallei<\/jats:italic>, and <jats:italic>Yersinia pestis<\/jats:italic>) inactivated with H<jats:sub>2<\/jats:sub>O<jats:sub>2<\/jats:sub> vapors or by heat treatment at 62.5\u00b0C for 24\u00a0h. The DF\u2010IEF chip method continually separated and concentrated the inactivated bacterial cells for subsequent detection using MALDI\u2010TOF MS. The content of the inactivated bacteria in the DF\u2010IEF chip fractions was evaluated with the MS analysis, where inactivated <jats:italic>Y. pestis<\/jats:italic> was found to be the most efficiently focusing species. Sensitivity analysis showed limits as low as 2\u00a0\u00d7\u00a010<jats:sup>5<\/jats:sup> colony forming units per mL for inactivated <jats:italic>B. anthracis<\/jats:italic>.<\/jats:p>","DOI":"10.1002\/elps.202400052","type":"journal-article","created":{"date-parts":[[2025,1,3]],"date-time":"2025-01-03T00:40:23Z","timestamp":1735864823000},"page":"212-220","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Isoelectric Focusing Fractionation Method for Signal Enhancement in Detection of Inactivated Biological Agents Using Matrix\u2010Assisted Laser Desorption\/Ionization Mass Spectrometry"],"prefix":"10.1002","volume":"46","author":[{"given":"Filip","family":"Du\u0161a","sequence":"first","affiliation":[{"name":"Institute of Analytical Chemistry of the Czech Academy of Sciences  Brno Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ji\u0159\u00ed","family":"\u0160alplachta","sequence":"additional","affiliation":[{"name":"Institute of Analytical Chemistry of the Czech Academy of Sciences  Brno Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marie","family":"Hork\u00e1","sequence":"additional","affiliation":[{"name":"Institute of Analytical Chemistry of the Czech Academy of Sciences  Brno Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kamila","family":"Lunerov\u00e1","sequence":"additional","affiliation":[{"name":"National Institute for Nuclear, Chemical and Biological Protection  Kamenna Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Veronika","family":"\u010cerm\u00e1kov\u00e1","sequence":"additional","affiliation":[{"name":"National Institute for Nuclear, Chemical and Biological Protection  Kamenna Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michal","family":"D\u0159ev\u00ednek","sequence":"additional","affiliation":[{"name":"National Institute for Nuclear, Chemical and Biological Protection  Kamenna Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Old\u0159ich","family":"Kub\u00ed\u010dek","sequence":"additional","affiliation":[{"name":"National Institute for Nuclear, Chemical and Biological Protection  Kamenna Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,1,2]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2010.04.005"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.arabjc.2022.104521"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12275-018-7457-0"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-011-3845-7"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.meegid.2004.02.004"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00857-09"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.01724-13"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2021.339333"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhin.2022.05.006"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.02004-08"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens9050408"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2023.116947"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2023.117018"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201200136"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2017.08.046"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/0021-9673(93)E0836-J"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/0021-9673(94)01124-W"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2019.05.010"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2022.340035"},{"key":"e_1_2_9_21_1","first-page":"21","article-title":"EU Directive 2000\/54\/EC, Protection of Workers From Risks Related to Exposure to Biological Agents at Work","volume":"262","author":"E. 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