Comparative mass spectrometry analysis of high and low centrifugation extracellular vesicle (EV) pellets from healthy urine following Tamm-Horsfall protein removal.

Urinary extracellular vesicles (uEVs) are potential biomarkers for renal physiology and disease. Their isolation is challenged by Tamm-Horsfall protein (THP) contamination, particularly in high-speed ultracentrifugation (P100) fractions. Low-speed (P20) fractions contain biologically relevant uEVs, yet their proteomic composition is underexplored. This study evaluates strategies for THP removal to optimize mass spectrometry analysis and characterizes P20 and P100 uEV proteomes and its combination in healthy urine. Urine from a healthy volunteer was processed via sequential centrifugation at 21,200 g (P20) and 164,244 g (P100). THP removal was performed using low ionic strength buffer (P20) and size-exclusion chromatography (P100). Western blotting, nanoparticle tracking, and mass spectrometry were used to characterize protein content. Functional analysis was performed to assess biological significance. Mass spectrometry identified 1,103 unique proteins in P100, 929 in P20, and 1,676 in the combined pellet, with 90% similarity between fractions. P20 was enriched in proteasome components (PSMC2, PSMD2, PSMD5, PSMD6, PSMD8, PSMD11, PSMD13, PSME1), while P100 had no advantage in terms of expression profile in any of the proteins examined. Functional analysis indicated immune activation in P100 and protein degradation in P20, suggesting complementary biological roles. This study reveals proteasome enrichment in P20 uEVs and supports a combined P20 + P100 approach to improve biomarker discovery. Optimized THP removal strategies enhance mass spectrometry resolution, facilitating uEV analysis for clinical and research applications. INSTITUTIONAL REVIEW BOARD FOR HEALTH SCIENCES RESEARCH (IRB-HSR) AT UVA: HSR220053.