Blood extracellular vesicles (EVs) play essential roles in cell–cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC–MS/MS and expand the proteome coverage. Plasma depletion was performed using a single-use spin column and EVs were isolated from only 100 µL of non-depleted and depleted plasma by size exclusion chromatography. Afterwards, EVs were characterized by nanoparticle tracking analysis and mass spectrometry–based proteomics using a data-independent acquisition approach. Depleted plasma-derived EVs had higher particle concentrations and particle-to-protein ratios. Depletion did increase the protein coverage with a higher number of identifications in EVs from depleted plasma (474 proteins) than from non-depleted (386 proteins). However, EVs derived from non-depleted plasma carried a slightly higher number of common EV markers. Overall, our findings suggest that plasma depletion prior to EV isolation by size exclusion chromatography provides higher yield and protein coverage, but slightly lower identification of EV markers. This study also showed the possibility to characterize the proteome of EVs derived from small plasma volumes, encouraging the clinical feasibility of the discovery of EV biomarkers.

Extracellular vesicles (EVs) form a heterogeneous group of membrane-enclosed structures secreted by all cell types. EVs export encapsulated materials composed of proteins, lipids, and nucleic acids, making them a key mediator in cell–cell communication. In the context of the neurovascular unit (NVU), a tightly interacting multicellular brain complex, EVs play a role in intercellular communication and in maintaining NVU functionality. In addition, NVU-derived EVs can also impact peripheral tissues by crossing the blood–brain barrier (BBB) to reach the blood stream. As such, EVs have been shown to be involved in the physiopathology of numerous neurological diseases. The presence of NVU-released EVs in the systemic circulation offers an opportunity to discover new diagnostic and prognostic markers for those diseases. This review outlines the most recent studies reporting the role of NVU-derived EVs in physiological and pathological mechanisms of the NVU, focusing on neuroinflammation and neurodegenerative diseases. Then, the clinical application of EVs-containing molecules as biomarkers in acute brain injuries, such as stroke and traumatic brain injuries (TBI), is discussed.

Proteomic and transcriptomic analyses of cerebrospinal fluid (CSF)‐derived extracellular vesicles (EVs) offer unique insights into molecular changes associated with central nervous system (CNS) diseases and may result in biomarker identification. No gold standard method to enrich EVs from CSF has been established, and head‐to‐head comparisons of outputs of different protocols are scarce. Using a large pool of CSF, we characterised the EV preparations resulting from four enrichment protocols and compared them in terms of yield and purity. We found that particles enriched by ultracentrifugation (UC) or a combination of ultrafiltration and size exclusion chromatography (UF‐SEC) exhibited the typical morphological and biochemical characteristics of small EVs and were highly enriched in proteins and polyadenylated (polyA) transcripts associated with EV‐related biological processes. UF‐SEC preparations had higher particle yields, whilst more proteins were identified in UC preparations. Approximately 40% of the EV preparations’ proteome was not identified in unenriched CSF, among which a core proteome of 45 proteins was identified in 30 EV preparations from independent experiments, which may serve as CSF‐derived EV markers. Enrichment scores to protein contaminants, albumin and apolipoprotein E were higher in UF‐SEC preparations. In conclusion, all protocols analysed here resulted in enrichment of particles with small EV characteristics, with EV enrichments from UF‐SEC resulting in the highest yield and purity.

Managing acute stroke is challenging and requires the differentiation of stroke subtypes while excluding stroke mimics. Understanding the biological processes underlying the different stroke subtypes could help improve acute stroke care and patient outcomes. Plasma-derived extracellular vesicles (EVs) have emerged as promising tools for investigating these processes through their unique cargo. Therefore, we aimed at exploring the protein content of plasma-derived EVs in a cohort composed of patients with hemorrhagic stroke (n = 10), ischemic stroke with large-vessel occlusion (LVO) (n = 10) and without LVO (n = 10), transient ischemic attack (n = 10), stroke mimics (n = 10) and healthy controls (n = 10). EVs were isolated by size exclusion chromatography from 150 μL of plasma. Characterization was performed by transmission electron microscopy, western blotting and nanoparticle tracking analysis, and confirmed an efficient EV enrichment. Proteomics analysis was conducted using data-independent acquisition mass spectrometry and a supervised partial least squares discriminant analysis (PLS-DA) model was applied on proteomics data. The PLS-DA model successfully distinguished healthy controls, severe stroke subtypes (LVO ischemic and hemorrhagic stroke), and non LVO ischemic stroke, identifying key proteins influencing these patterns. These findings suggest that EVs and their protein cargo may play a role in critical processes like inflammation and coagulation in acute stroke pathology.