UFLC-derived CSF extracellular vesicle origin and proteome.

Cerebrospinal fluid (CSF) extracellular vesicles (EVs) show promise as a source of neurological disease biomarkers, though their precise origin is poorly understood. Current extraction techniques produce disappointing yield and purity. This study describes the application of ultrafiltration liquid chromatography (UFLC) to CSF-EVs, compared with ultracentrifugation (UC), and explores CSF-EV origin. EVs were extracted from human CSF by UC and UFLC and characterized using nanoparticle tracking analysis, electron microscopy and immunoblotting. EV and CSF proteomes were analysed by LC-MS/MS. UFLC-isolated particles have size, morphology and marker expression characteristic of EVs. UFLC provides greater EV yield (UFLC 7.90 × 108 ± SD 1.31 × 108 EVs/mL CSF, UC 1.06 × 108 ±0.57 × 108 p<0.001). UFLC enhances purity, proteomic depth (UFLC 704±52, UC 340±57 identifications, p<0.01) and consistency of quantification (CV 17% vs 23%). EVs contain more intracellular proteins (OR 2.63 p<0.001) and fewer plasma proteins than CSF (OR 0.60, p<0.001). CSF and EV-enriched proteomes show overrepresentation of brain-specific proteins (EV OR 3.18, p<0.001; CSF OR 3.37, p<0.001). Overrepresentation of cerebral white matter (OR 1.99, p = 0.015) and choroid plexus proteins (OR 1.87, p<0.001) is observed in EVs. UFLC provides improves yield and purity of CSF-EVs. The EV-enriched proteome better reflects the intracellular and white matter proteome than whole CSF. This article is protected by copyright. All rights reserved.