Background: it is now established that Extracellular Vesicles (EVs) display potent regulatory functions in biological systems. Circulating EVs can enable extreme reduction of the high dynamic range of detectable protein concentration in whole biofluids, paving the way for the biomarker discovery in the so-called "dark proteome". The best strategy of EVs isolation from biofluids remains an open question especially considering their heterogeneity in terms of phenotype and their suitability for proteomic application, once purified. Within such a complex and dynamic scenario, we have recently optimized an innovative "FACS-Proteomics" workflow for the isolation and subsequent proteomics characterization of EVs directly from untouched biological fluids taking advantage of a lipophilic cationic dye (LCD) able to probe intact EVs. This purification method offers the great opportunity to carry out proteomics on circulating and specific EV populations by sub-typing them with an appropriate panel of antibodies.

Methods: FACS strategy was used to purify Leukocyte-derived EVs (Leuko EVs, LCD+/CD45+/Phalloidin- events) from blood and tears of healthy volunteers and patients affected by Multiple Sclerosis (MuS). After purification, Atomic Force Microscopy and Nanoparticle Tracking Analysis were used to investigate Leuko EVs size distribution, morphology and number in both blood and tears extracts. Label free proteomics approach was carried out by NanoLC-Orbitrap-Fusion-Tribrid Mass Spectrometer; raw data were processed by MaxQuant and loaded on Ingenuity Pathway Analysis (IPA) for functional investigation.

Results: FACS isolated CD45+ EVs from blood and tears (1.4x105 and 2x106 events, respectively) displayed a high level of purity (>90%) and more than 90% of proteins (78 in tear and 155 blood) identified in both biological fluids were mainly referred to the leukocyte phenotype. The same workflow was applied to carry out proteomics on purified Leuko EVs from tears of Multiple Sclerosis (MuS) patients, demonstrating that they were enriched with an encoded protein cargo, reflecting the neuroinflammatory condition of MuS together with an increase of vascular networks (AGNPT2 and VEGF, upstream regulators activated) which reflects pro-angiogenic processes predicted as downstream effects. As leuko-EVs are still a heterogeneous group, we investigated it further, analysing the protein cargo of EVs released from neutrophils (the most abundant sub-group of white cells) and their activated counterpart the polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) (LOX1±), that in tourn seem to act in counteracting neuroinflammation although their role is still controversial. Our data showed that the combined FACS-Proteomics strategy for EV single phenotype characterization could have groundbreaking avenue for biomarker discovery, exalting the clinical value of tears EVs and helping in a better understanding of the EV-mediated processes in vivo.