The Importance of Extracellular Vesicle Purification for Downstream Analysis: A Comparison of Differential Centrifugation and Size Exclusion Chromatography for Parasitic Helminths

ABSTRACT: Fascioliasis is a major neglected tropical disease that infects humans and ruminant species worldwide. Recent discoveries have identified that liver fluke extracellular vesicles are released into the host’s internal environment. To understand extracellular vesicle biology further, isolation of the extracellular vesicle is necessary for downstream applications. The most widely used purification method involves differential centrifugation however; there is no ‘gold standard’ to date of extracellular vesicle isolation. Recently, size exclusion chromatography has been used to successfully purify EVs. In this study, liver flukes were collected to compare size exclusion chromatography EV purification, to the widely accepted method of differential centrifugation. Transmission electron microscopy and atomic force microscopy showed that size exclusion chromatography extracellular vesicles were smaller in size and diversity than differential centrifugation purified extracellular vesicles. Protein concentration and western blotting suggested that size exclusion chromatography purification had a high EV purity to protein yield ratio in comparison to differential centrifugation. Mass spectrometry analysis discovered a greater amount of protein identifications and unique peptide hits in differential centrifugation purified EVs with less diverse functionality, than size exclusion chromatography purified EVs. However, gene enrichment analysis against the Fasciola hepatica genome showed comparable results between purification methods. Furthermore, western blotting revealed that glutathione s-transferase sigma could be a potential marker for liver fluke EVs. These results suggest that size exclusion chromatography purification produces a higher EV purity to protein yield ratio and maintains extracellular vesicle composition, so therefore is a better suited method for further downstream experimentation with liver fluke extracellular vesicles, compared to differential centrifugation.

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Fasciola Hepatica (liver Fluke)

SUBMITTER: Chelsea Davis  

LAB HEAD: Prof. Peter Brophy

PROVIDER: PXD008737 | Pride | 2019-02-13

REPOSITORIES: Pride