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Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS an...

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Autores principales: Askeland, Anders, Borup, Anne, Østergaard, Ole, Olsen, Jesper V., Lund, Sigrid M., Christiansen, Gunna, Kristensen, Søren R., Heegaard, Niels H. H., Pedersen, Shona
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459681/
https://www.ncbi.nlm.nih.gov/pubmed/32722497
http://dx.doi.org/10.3390/biomedicines8080246
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author Askeland, Anders
Borup, Anne
Østergaard, Ole
Olsen, Jesper V.
Lund, Sigrid M.
Christiansen, Gunna
Kristensen, Søren R.
Heegaard, Niels H. H.
Pedersen, Shona
author_facet Askeland, Anders
Borup, Anne
Østergaard, Ole
Olsen, Jesper V.
Lund, Sigrid M.
Christiansen, Gunna
Kristensen, Søren R.
Heegaard, Niels H. H.
Pedersen, Shona
author_sort Askeland, Anders
collection PubMed
description Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.
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spelling pubmed-74596812020-09-02 Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation Askeland, Anders Borup, Anne Østergaard, Ole Olsen, Jesper V. Lund, Sigrid M. Christiansen, Gunna Kristensen, Søren R. Heegaard, Niels H. H. Pedersen, Shona Biomedicines Article Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both. MDPI 2020-07-25 /pmc/articles/PMC7459681/ /pubmed/32722497 http://dx.doi.org/10.3390/biomedicines8080246 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Askeland, Anders
Borup, Anne
Østergaard, Ole
Olsen, Jesper V.
Lund, Sigrid M.
Christiansen, Gunna
Kristensen, Søren R.
Heegaard, Niels H. H.
Pedersen, Shona
Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation
title Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation
title_full Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation
title_fullStr Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation
title_full_unstemmed Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation
title_short Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation
title_sort mass-spectrometry based proteome comparison of extracellular vesicle isolation methods: comparison of me-kit, size-exclusion chromatography, and high-speed centrifugation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459681/
https://www.ncbi.nlm.nih.gov/pubmed/32722497
http://dx.doi.org/10.3390/biomedicines8080246
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