Ultrafiltration combing with phospholipid affinity-based isolation for metabolomic profiling of urinary extracellular...

Recent years have seen the field of extracellular vesicle (EV) studies burgeoning. This is mainly because EV constituents including nucleic acid, proteins, lipids, and metabolites are promising sources towards disease biomarker discovery. However, EV study remains challenging due to the complexity of biofluids as well as technical limitations during sample preparation. Here, we proposed a simple method combing ultrafiltration (UF) and phospholipid affinity to collect high purity EVs from 30 mL of urine sample for their metabolomic profiling. Ultracentrifugation (UC) for EV isolation was applied as a reference method. Western blot (WB) analysis, nanoparticles tracking analysis (NTA) and electron microscopy (EM) were used to assess EV protein markers and to characterize vesicle size and morphology. The results revealed that more than 10¹⁰ EV particles could be isolated from a 30 mL urine sample by the proposed method, and the resulting EVs carry specific protein markers and had a typical "cup shape" morphology. This suggests that our method is suitable for EV isolation and can provide sufficient EV quantity to ensure downstream analysis. Further untargeted metabolomic profiling of isolated EVs by UHPLC-QTOF-MS detected 433 metabolites by our methods and 432 metabolites by UC with a MS/MS similarity score greater than 0.7. We then applied the lipid metabolites-targeted method using UHPLC-QTrap-MS with the MRM mode, which successfully detected 467 compounds from urine EVs. This indicates that UF integrating phospholipid affinity is a reliable method for metabolic analysis of urinary EVs, which holds the potential for EV clinical application towards biomarker investigation from their metabolites.