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Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues
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Comparing the Proteomic Profiles of Extracellular Vesicles Isolated using Different Methods from Long-term Stored
Ana Torres1,2, Lorena Bernardo1,2, Carmen Sánchez1,2
1WHO Collaborating Centre for Leishmaniasis, Spanish National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Spain.
Biological Procedures Online
|June 19, 2024
Summary
Combining size exclusion chromatography and ultracentrifugation (SEC+UC) enhances extracellular vesicle (EV) purity from stored plasma. This improved EV isolation method enables comprehensive proteomic analysis for novel biomarker discovery.
Area of Science:
- Biochemistry
- Biotechnology
- Proteomics
Background:
- Standardized extracellular vesicle (EV) isolation protocols are lacking, particularly for biobank-stored plasma.
- This limits the discovery and study of novel EV biomarkers for clinical applications.
Purpose of the Study:
- To evaluate a combined SEC+UC method for isolating highly pure EVs from stored plasma.
- To assess the suitability of SEC+UC-isolated EVs for comprehensive proteomic analysis and biomarker discovery.
Main Methods:
- EVs were isolated from human plasma using ultracentrifugation (UC), size exclusion chromatography (SEC), or a combined SEC+UC approach.
- EV characterization involved transmission electron microscopy, nanoparticle tracking analysis, western blotting, and proteomic analysis.
Main Results:
- The SEC+UC method yielded EVs with higher purity and a more complex proteome compared to UC or SEC alone.
- Western blotting indicated reduced plasma protein contamination (albumin) in SEC+UC isolates.
- Proteomic analysis of SEC+UC isolates identified 181 unique proteins, including low-abundance plasma proteins, suitable for biomarker discovery.
Conclusions:
- The SEC+UC isolation strategy provides highly pure and diverse EVs from biobank-stored plasma.
- This method is suitable for in-depth proteomic analysis, facilitating the identification of novel biomarkers for diagnostics and personalized medicine.
