Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Gustavo Diaz; Chandler Bridges; Megan Lucas; Yong Cheng; Jeff S. Schorey; Karen M. Dobos; Nicole A. Kruh-Garcia

doi:10.3791/57467

Method Article

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

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DOI:

10.3791/57467

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April 13th, 2018

Gustavo Diaz¹ , Chandler Bridges¹ , Megan Lucas¹ , Yong Cheng² , Jeff S. Schorey² , Karen M. Dobos¹ , Nicole A. Kruh-Garcia¹

¹Department of Microbiology, Immunology and Pathology, Colorado State University, ²Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame

Summary

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Abstract

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Introduction

Exosomes are nanovesicles (ranging in size from 30 nm to 150 nm) released by almost all the cells in the human body to facilitate cell-to-cell communication processes¹^,². Interestingly, the composition of exosomes changes depending on the cells of origin as well as the health status of the individual³^,⁴^,⁵. Additionally, exosomes can be retrieved from several biological fluids such as: saliva, urine, and blood². Because of these features, exosomes are considered to be a good source of disease bi....