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Updated: Oct 29, 2025
Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum
Published on: April 13, 2018
Coupling size exclusion chromatography to ultracentrifugation improves detection of exosomal proteins from human
Sara Alameldin1,2, Victor Costina1, Hesham A Abdel-Baset2
1Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany.
Objectives:
Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30-140 nm. They are constantly produced by different cell types, by both healthy and abnormal cells, and can be isolated from almost all body fluids.Little information exists in isolating exosomes from plasma due to the complexity of its content and the presence of contaminating plasma proteins.
Design And Methods:
We carried-out liquid chromatography-mass spectrometry (LC-MS/MS) analyses of plasma-derived vesicles from 4 healthy donors obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes.We compared the proteins detected by UC alone and UC coupled with SEC.
Results:
In the coupled UC + SEC methodology we found 52.25% more proteins enriched in exosomes as CD9, Annexins, YWHAZ (14-3-3 family) and others, than by using UC alone. There is also a reduction of 98.8% of contaminating plasma proteins by coupling UC and SEC in comparison to using UC alone.
Conclusions:
We conclude that exosomes can be successfully isolated from plasma using a very simple combination of standard methods, which could largely improve the proteomics profiling of plasma exosomes.
Insights
Researchers successfully isolated exosomes from plasma using ultracentrifugation and size-exclusion chromatography. This combined method significantly enriches exosome proteins and reduces contaminants for better proteomic analysis.
Area of Science:
- Extracellular Vesicles Biology
- Proteomics
- Biochemistry
Background:
- Exosomes are nanoscale vesicles originating from endocytosis, present in various body fluids.
- Isolation of exosomes from plasma is challenging due to complex composition and protein contamination.
- Limited information exists on effective plasma exosome isolation techniques.
Purpose of the Study:
- To develop and evaluate a method for isolating and enriching exosomes from human plasma.
- To compare the efficacy of combined ultracentrifugation (UC) and size-exclusion chromatography (SEC) with UC alone for plasma exosome isolation.
- To assess the impact of the isolation method on exosome protein profiling.
Main Methods:
- Plasma samples from 4 healthy donors were analyzed.
- Exosomes were isolated using ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC).
- Liquid chromatography-mass spectrometry (LC-MS/MS) was employed to compare protein content.
Main Results:
- The coupled UC + SEC method identified 52.25% more exosomal proteins (e.g., CD9, Annexins, YWHAZ) compared to UC alone.
- A significant 98.8% reduction in contaminating plasma proteins was achieved with the combined UC + SEC approach.
- The enhanced isolation method improved the yield and purity of plasma exosomes.
Conclusions:
- A simple combination of UC and SEC effectively isolates exosomes from plasma.
- This method significantly enhances exosome protein enrichment and reduces contaminants.
- The developed technique holds promise for improving plasma exosome proteomics profiling.
