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Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

Journal article
Authors Nasibeh Karimi
Aleksander Cvjetkovic
Su Chul Jang
Rossella Crescitelli
M. A. H. Feizi
R. Nieuwland
Jan Lötvall
Cecilia Lässer
Published in Cellular and Molecular Life Sciences
Volume 75
Issue 15
Pages 2873-2886
ISSN 1420-682X
Publication year 2018
Published at Krefting Research Centre
Institute of Medicine, Department of Internal Medicine and Clinical Nutrition
Pages 2873-2886
Language en
Keywords Exosomes, Extracellular vesicles, Lipoproteins, Plasma, Serum, Size-exclusion chromatography, Density cushion, Mass spectrometry, Proteomics, blood-plasma, exosomes, microvesicles, cells, density, rna, chromatography, subpopulations, micrornas, proteins, Biochemistry & Molecular Biology, Cell Biology
Subject categories Clinical Medicine


The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8-12, while the bulk of the plasma proteins was present in fractions 11-28. Vesicle markers peaked in fractions 7-11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.

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