Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in‐depth proteomic analysis

• Published in: Journal of extracellular vesicles Vol. 11; no. 2; pp. e12184 - n/a
• Main Authors: Correll, Vanessa L., Otto, Joseph J., Risi, Cristina M., Main, Brian P., Boutros, Paul C., Kislinger, Thomas, Galkin, Vitold E., Nyalwidhe, Julius O., Semmes, O. John, Yang, Lifang
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.02.2022; John Wiley and Sons Inc; Wiley
• Subjects: Biomarkers; Biopsy; Contaminants; Disease; Dithiothreitol; EPS‐urine; expressed prostatic secretions in urine; extracellular vesicle; Extracellular vesicles; Extracellular Vesicles - chemistry; Humans; Male; Mass Spectrometry; Mass spectroscopy; Patients; Polymers; Prostate; Prostate cancer; Protein purification; Proteins; Proteomics; Proteomics - methods; Secretions; Surveillance; Tamm‐Horsfall protein; Ultracentrifugation; Urine; uromodulin; United States > US; Virginia; prostate cancer; Tamm-Horsfall protein; proteomics; urine; biomarkers; mass spectrometry; uromodulin; EPS-urine; extracellular vesicle; expressed prostatic secretions in urine
• ISSN: 2001-3078; 2001-3078
• DOI: 10.1002/jev2.12184

The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co‐isolate protein contaminants that mask lower‐abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm‐Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co‐isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer‐associated EV‐resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs.