title: Ready-made chromatography columns for extracellular vesicle isolation from plasma.
abstract: Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc.) that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio). The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma.
authors.affiliation: Institute of Cancer and Genetics, School of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff, United Kingdom.
authors.forename: Joanne Louise
authors.initials: JL
authors.lastname: Welton
authors.affiliation: Institute of Cancer and Genetics, School of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff, United Kingdom.
authors.forename: Jason Paul
authors.initials: JP
authors.lastname: Webber
authors.affiliation: Cell Guidance Systems, Cambridge, United Kingdom.
authors.forename: Laur-Alexandru
authors.initials: LA
authors.lastname: Botos
authors.affiliation: Cell Guidance Systems, Cambridge, United Kingdom.
authors.forename: Michael
authors.initials: M
authors.lastname: Jones
authors.affiliation: Institute of Cancer and Genetics, School of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff, United Kingdom; claytona@Cardiff.ac.uk.
authors.forename: Aled
authors.initials: A
authors.lastname: Clayton
keywords: chromatography; exosome precipitation; exosomes; nanoparticle tracking; plasma; ultracentrifugation
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