The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens.
BACKGROUND: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 centrif...
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ftdoajarticles:oai:doaj.org/article:74d755352dd04cf0b1bee8b916572f77 2023-05-15T15:12:41+02:00 The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. Chelsea N Davis Helen Phillips John J Tomes Martin T Swain Toby J Wilkinson Peter M Brophy Russell M Morphew 2019-02-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0007191 https://doaj.org/article/74d755352dd04cf0b1bee8b916572f77 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC6411213?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0007191 https://doaj.org/article/74d755352dd04cf0b1bee8b916572f77 PLoS Neglected Tropical Diseases, Vol 13, Iss 2, p e0007191 (2019) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2019 ftdoajarticles https://doi.org/10.1371/journal.pntd.0007191 2022-12-31T11:12:37Z BACKGROUND: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. METHODOLOGY/PRINCIPAL FINDINGS: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 diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs. CONCLUSIONS/SIGNIFICANCE:The data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 13 2 e0007191 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Chelsea N Davis Helen Phillips John J Tomes Martin T Swain Toby J Wilkinson Peter M Brophy Russell M Morphew The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
BACKGROUND: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. METHODOLOGY/PRINCIPAL FINDINGS: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 diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs. CONCLUSIONS/SIGNIFICANCE:The data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies. |
format |
Article in Journal/Newspaper |
author |
Chelsea N Davis Helen Phillips John J Tomes Martin T Swain Toby J Wilkinson Peter M Brophy Russell M Morphew |
author_facet |
Chelsea N Davis Helen Phillips John J Tomes Martin T Swain Toby J Wilkinson Peter M Brophy Russell M Morphew |
author_sort |
Chelsea N Davis |
title |
The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. |
title_short |
The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. |
title_full |
The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. |
title_fullStr |
The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. |
title_full_unstemmed |
The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. |
title_sort |
importance of extracellular vesicle purification for downstream analysis: a comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2019 |
url |
https://doi.org/10.1371/journal.pntd.0007191 https://doaj.org/article/74d755352dd04cf0b1bee8b916572f77 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 13, Iss 2, p e0007191 (2019) |
op_relation |
http://europepmc.org/articles/PMC6411213?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0007191 https://doaj.org/article/74d755352dd04cf0b1bee8b916572f77 |
op_doi |
https://doi.org/10.1371/journal.pntd.0007191 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
13 |
container_issue |
2 |
container_start_page |
e0007191 |
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1766343343983296512 |