The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens

PLoS Neglected Tropical Diseases
Chelsea N DavisRussell M Morphew

Abstract

Robust protocols for the isolation of extracellular vesicles (EVs) from the rest of their excretory-secretory products are necessary for downstream studies and application development. The most widely used purification method of EVs for helminth pathogens is currently differential centrifugation (DC). In contrast, size exclusion chromatography (SEC) has been included in the purification pipeline for EVs from other pathogens, highlighting there is not an agreed research community 'gold standard' for EV isolation. In this case study, Fasciola hepatica from natural populations were cultured in order to collect EVs from culture media and evaluate a SEC or DC approach to pathogen helminth EV purification. Transmission electron and atomic force microscopy demonstrated that EVs prepared by SEC were both smaller in size and less diverse than EV resolved by DC. Protein quantification and Western blotting further demonstrated that SEC purification realised a higher EV purity to free excretory-secretory protein (ESP) yield ratio compared to DC approaches as evident by the reduction of soluble free cathepsin L proteases in SEC EV preparations. Proteomic analysis further highlighted DC contamination from ESP as shown by an increased diversi...Continue Reading

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Citations

Sep 13, 2019·Journal of Neuroimmune Pharmacology : the Official Journal of the Society on NeuroImmune Pharmacology·Ryan P McNamara, Dirk P Dittmer
Sep 19, 2020·International Journal of Molecular Sciences·Claudia CavallariMaria Felice Brizzi
Oct 2, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Jing LiChenxi Yang
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Jan 14, 2021·Molecular Immunology·Karen J MontañoJavier Sotillo
Mar 21, 2021·Analytical Biochemistry·Teran CloetHuiyan Li
Apr 4, 2021·Pathogens·Adam P S Bennett, Mark W Robinson
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Jun 13, 2020·International Journal for Parasitology·Christian M Sánchez-LópezDolores Bernal
Jul 12, 2020·International Journal for Parasitology·Javier SotilloLucienne Tritten
Aug 7, 2021·Pathogens·Boontarikaan WititkornkulRussell M Morphew

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Datasets Mentioned

BETA
PXD008737
AAB41670.2
AAF44676.1
ACJ12893.1

Methods Mentioned

BETA
size exclusion chromatography
electrophoresis
Chip
atomic
transmission electron microscopy
AFM
atomic force microscopy
GTPase

Software Mentioned

imageJ
R
BLAST2GO
GOATOOLS
MASCOT
R package
Gwyddion
BLAST
Mass Hunter Qualitative Analysis

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