ORCID

0000-0001-9711-5768 (Yang)

Document Type

Article

Publication Date

2026

DOI

10.1186/s12014-026-09615-2

Publication Title

Clinical Proteomics

Volume

Advance online publication

Pages

34 pp.

Abstract

Objectives

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.

Methods

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.

Results

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.

Conclusion

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.

Rights

© The Authors 2026.

This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original authors and the source, provide a link to the Creative Commons license, and indicate if you modified the licensed material. You do not have permission under this license to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

Original Publication Citation

Bielopolski, D., Musante, L., Molina, H., Barrows, D., Upson, S., Yang, L., Carrol, T., Tobin, J. N., Kost, R. G., & Erdbrügger, U. (2026). Comparative mass spectrometry analysis of high and low centrifugation extracellular vesicle (EV) pellets from healthy urine following Tamm-Horsfall protein removal. Clinical Proteomics. Advance online publication. https://doi.org/10.1186/s12014-026-09615-2

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