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

Journal of Extracellular Vesicles

Thomas Kislinger¹  Paul C. Boutros¹  Cristina M. Risi²  Vitold E. Galkin²  Joseph J. Otto³  Lifang Yang³  Brian P. Main³  Julius O. Nyalwidhe³  Vanessa L. Correll³  O. John Semmes³ 

[1] Department of Medical Biophysics University of Toronto Toronto Canada;Department of Physiological Sciences Eastern Virginia Medical School Norfolk Virginia USA;Leroy T. Canoles Jr. Cancer Research Center Eastern Virginia Medical School Norfolk Virginia USA;

关键词: biomarkers;    EPS‐urine;    expressed prostatic secretions in urine;    extracellular vesicle;    mass spectrometry;    prostate cancer;   

DOI  :  10.1002/jev2.12184

来源: DOAJ

【 摘 要 】

Abstract 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.

【 授权许可】

Unknown