{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T18:17:45Z","timestamp":1792174665992,"version":"4.4.0"},"reference-count":29,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T00:00:00Z","timestamp":1787961600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T00:00:00Z","timestamp":1787961600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100004191","name":"Novo Nordisk","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004191","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["isevjournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J of Extracellular Bio"],"published-print":{"date-parts":[[2026,9]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Extracellular vesicles (EVs) hold significant promise as biomarkers, but their clinical application is constrained by variability in pre\u2010analytical handling and isolation. EV isolation methods directly shape which plasma\u2010derived EV\u2010containing preparations are captured, yet systematic method comparisons across multiple analytical dimensions are limited. We comprehensively evaluated eleven EV isolation methods in pooled platelet\u2010poor plasma (5 donors; 6 technical replicates\/method). We further evaluated selected methods using five individual donors. EVs were quantified by NanoFCM, profiled for tetraspanins (CD9, CD63, CD81) via MSD assays, and further characterized by LC\u2010MS\/MS proteomics. We show that different EV isolation methods for plasma produce different EV containing preparation. EV isolation methods broadened proteome coverage in plasma but showed divergent performance. While all methods captured EVs in the 50\u2013150\u00a0nm range, centrifugation and ultracentrifugation identified the broadest proteomes (up to 1093 proteins) driven by higher plasma protein carryover. Conversely, ExoEasy and qEV 70 isolated larger EVs and achieved stronger depletion of abundant plasma proteins but showed lower proteome coverage. A total of 117 proteins were detected across all isolation methods. Pre\u2010clearing of samples removed contaminants but at the cost of protein identifications. We demonstrate that method selection must align with the specific analytical goal: centrifugation for comprehensive proteome profiling, affinity\/size\u2010exclusion methods for contaminant\u2010sensitive assays, and precipitation for high\u2010throughput applications. This systematic characterization provides an evidence\u2010based framework and look\u2010up resource for matching isolation strategies to downstream applications and research questions.<\/jats:p>","DOI":"10.1002\/jex2.70180","type":"journal-article","created":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T18:32:42Z","timestamp":1788028362000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Method Matters: A Proteomics\u2010Informed Framework for Selecting Extracellular Vesicle Isolation Methods for Plasma"],"prefix":"10.1002","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-2262-7587","authenticated-orcid":false,"given":"Scheila Julia","family":"Werle","sequence":"first","affiliation":[{"name":"Novo Nordisk A\/S  M\u00e5l\u00f8v Denmark"},{"name":"Gastro Unit Copenhagen University Hospital, Amager and Hvidovre Hospital  Hvidovre Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1198-8619","authenticated-orcid":false,"given":"Marie Louise Nautrup","family":"Therkelsen","sequence":"additional","affiliation":[{"name":"Novo Nordisk A\/S  M\u00e5l\u00f8v Denmark"},{"name":"Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5968-6719","authenticated-orcid":false,"given":"Chen","family":"Meng","sequence":"additional","affiliation":[{"name":"Novo Nordisk A\/S  M\u00e5l\u00f8v Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4011-6406","authenticated-orcid":false,"given":"Mads","family":"Gr\u00f8nborg","sequence":"additional","affiliation":[{"name":"Novo Nordisk A\/S  M\u00e5l\u00f8v Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9462-4468","authenticated-orcid":false,"given":"Lise Lotte","family":"Gluud","sequence":"additional","affiliation":[{"name":"Gastro Unit Copenhagen University Hospital, Amager and Hvidovre Hospital  Hvidovre Denmark"},{"name":"Department of Clinical Medicine, Faculty of Health and Medical Sciences Copenhagen University  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2733-8541","authenticated-orcid":false,"given":"Dres","family":"Damgaard","sequence":"additional","affiliation":[{"name":"Novo Nordisk A\/S  M\u00e5l\u00f8v Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,29]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-57497-7"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12951-022-01256-5"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.3390\/cells8070727"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.9b04480"},{"key":"e_1_2_9_6_1","doi-asserted-by":"crossref","unstructured":"Figueiras R. S.Vagueiro R.Kay et\u00a0al.2025. \u201cOrgan\u2010Specific Isolation of Hepatocyte Extracellular Vesicles from Human Plasma Enables Tissue\u2010Resolved Proteomic and miRNA Profiling.\u201dbioRxiv.","DOI":"10.1101\/2025.06.13.658908"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms221910510"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2019.1621131"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.3402\/jev.v3.26913"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21207568"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201300282"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1615\/ForumImmunDisTher.2016016491"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.202200039"},{"issue":"8","key":"e_1_2_9_14_1","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0236439","article-title":"Extracellular Vesicles From Human Plasma and Serum Are Carriers of Extravesicular Cargo\u2010Implications for Biomarker Discovery","volume":"15","author":"Mari P.","year":"2020","journal-title":"PLoS ONE"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.3390\/biology10050359"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2020.06.011"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.3402\/jev.v5.29975"},{"issue":"1","key":"e_1_2_9_18_1","first-page":"9","article-title":"Multiplex Electrochemiluminescence Immunoassays for Phenotyping of Intact Extracellular Vesicles (EVs)","volume":"96","author":"Routenberg D. A.","year":"2020","journal-title":"Meso Scale Discovery (MSD)"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2015.00050"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2020.07.014"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41420-020-00305-y"},{"key":"e_1_2_9_22_1","doi-asserted-by":"crossref","unstructured":"Ter\u2010Ovanesyan D. S.Whiteman T.Gilboa et\u00a0al.2024. \u201cIdentification of Markers for the Isolation of Neuron\u2010Specific Extracellular Vesicles.\u201dBioRxiv.","DOI":"10.1101\/2024.04.03.587267"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471143030.cb0322s30"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2018.1535750"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2017.1344087"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1039\/C5OB01451D"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41417-023-00617-y"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.3402\/jev.v4.27066"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2019.1697028"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.5c00149"}],"container-title":["Journal of Extracellular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/isevjournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jex2.70180","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/isevjournals.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/jex2.70180","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/isevjournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jex2.70180","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T05:43:48Z","timestamp":1789969428000},"score":1,"resource":{"primary":{"URL":"https:\/\/isevjournals.onlinelibrary.wiley.com\/doi\/10.1002\/jex2.70180"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,29]]},"references-count":29,"journal-issue":{"issue":"9","published-print":{"date-parts":[[2026,9]]}},"alternative-id":["10.1002\/jex2.70180"],"URL":"https:\/\/doi.org\/10.1002\/jex2.70180","archive":["Portico"],"relation":{},"ISSN":["2768-2811","2768-2811"],"issn-type":[{"value":"2768-2811","type":"print"},{"value":"2768-2811","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,29]]},"assertion":[{"value":"2026-03-26","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-23","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-29","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70180"}}