Feasibility of urinary extracellular vesicle proteome profiling using a robust and simple, clinically applicable...

Area of Science:

• Biochemistry
• Molecular Biology
• Urology

Background:

• Prostate cancer (PCa) diagnosis and prognosis can be improved using biomarkers.
• Urinary extracellular vesicles (EVs) are a promising source for non-invasive liquid biopsies.
• Current EV isolation methods can be time-consuming and require specialized equipment.

Purpose of the Study:

• To optimize a novel urinary EV isolation protocol using a Vn96-peptide method.
• To compare the efficiency and yield of the Vn96-peptide method with ultracentrifugation.
• To assess the clinical feasibility and proteomic depth of the Vn96-peptide EV isolation for PCa biomarker discovery.

Main Methods:

• Optimized urinary EV isolation using a heat shock protein-targeting Vn96-peptide.
• Compared Vn96-peptide isolation with gold-standard ultracentrifugation.
• Performed proteome profiling of isolated EVs using mass spectrometry-based proteomics (n=4 per method).
• Investigated the effect of NP40 detergent on EV isolation yield and purity.

Main Results:

• The Vn96-peptide method is significantly faster (≈1.5 h vs >3.5 h) and requires no special equipment, enhancing clinical feasibility.
• Proteomic analysis identified >3000 proteins, with >85% overlap between Vn96-peptide and ultracentrifugation methods.
• Addition of NP40 detergent to Vn96-peptide isolation increased EV marker (TSG101, PDCD6IP) levels, indicating higher EV yield and purity.

Conclusions:

• The Vn96-peptide-based urinary EV isolation is a clinically feasible, rapid, and efficient method.
• This protocol supports large-scale proteomic profiling of urinary EVs for PCa biomarker discovery.
• Optimized isolation enhances the potential of urinary EVs as a non-invasive liquid biopsy for prostate cancer.