FACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:40394633", "kind": "science-record", "revision": 2, "content_hash": "sha256:5ecd9d177d705e0372972712599b70878c80ce0a07227f8b3fd1fe0a9334b25e", "created_at": "2026-11-01T20:30:32Z", "updated_at": "2026-11-01T21:35:12Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "FACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.", "doi": "10.1186/s12967-025-06558-4", "pmid": "40394633", "authors": ["Maria Concetta Cufaro", "Paola Lanuti", "Domenico De Bellis", "Serena Veschi", "Anna Piro", "Antonella Fontana", "Alice Di Sebastiano", "Davide Brocco", "Pasquale Simeone", "Serena Pilato", "Reza M H Khorooshi", "Valentina Tomassini", "Marianna Gabriella Rispoli", "Luca Federici", "Ilaria Cicalini", "Damiana Pieragostino", "Piero Del Boccio"], "journal": "Journal of translational medicine", "year": 2025, "abstract": "BACKGROUND: The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis.\nRESULTS: Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers. Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+ EVs), reaching a high level of purity (> 90%). Purified Leuko EVs were characterized using atomic force microscopy, nanoparticle tracking, and shotgun proteomics analysis revealing a similar coded protein cargo in both biological fluids. Subsequently, the same workflow was applied to tears from Relapsing-Remitting Multiple Sclerosis (RRMS) patients, revealing a Leuko EVs protein cargo enrichment that reflects the neuroinflammatory condition characteristics of RRMS. This enrichment was evidenced by the activation of upstream regulators TGFB1 and NFE2L2, which are associated with inflammatory responses. Additionally, the analysis identified markers indicative of endothelial cell proliferation and the development of enhanced vascular networks, with AGNPT2 and VEGF emerging as activated upstream regulators. These findings indicate the complex interplay between inflammation and angiogenesis in RRMS.\nCONCLUSIONS: In conclusion, our combined FACS-Proteomics strategy offers a promising approach for biomarker discovery, analysing cell-specific EV phenotypes directly from untouched body fluids, advancing the clinical value of tears EVs and improving the understanding of EV-mediated processes in vivo. Data are available via ProteomeXchange with the identifier PXD049036 and in EV-TRACK knowledgebase with ID: EV240150.", "claims": [{"id": "claim-method", "statement": "Moreover, to define the differential proteins significantly expressed between pathologic group and control one, a univariate statistical analysis was performed with a p-value threshold of 0.05 visualizing results as Volcano Plot.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:40394633", "locator": "sections[13] \"Leukocyte-derived EVs proteomics characterization\" paragraph 3", "quote": "Moreover, to define the differential proteins significantly expressed between pathologic group and control one, a univariate statistical analysis was performed with a p-value threshold of 0.05 visualizing results as Volcano Plot."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD049036. 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