Abstract
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.