Microfiltration isolation of human urinary exosomes for characterization by MS

Purpose: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein-binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies. Experimental design: We characterized a microfiltration approach using a low protein binding, hydrophilized polyvinylidene difluoride membrane to easily and efficiently isolate urinary exosomes from fresh, room temperature or 4 degrees C urine, with a simultaneous depletion of abundant urinary proteins. Using LC-MS, immunoblot analysis, and electron microscopy methods, we demonstrate this method to isolate intact exosomes and thereby enrich for a low abundant urinary proteome. Results: In comparison to other standard methods of exosome isolation including ultracentrifugation and nanofiltration, we demonstrate equivalent enrichment of the exosome proteome with reduced co-purification of abundant urinary proteins. Conclusion and clinical relevance: In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput.