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

doi:10.3390/biomedicines8080246

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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

^1,*,†

^1,†,

^2,3,†,

³,

¹,

⁴,

^1,5,

^2,6,§ and

^1,5,*

Department of Clinical Biochemistry, Aalborg University Hospital, DK-9000 Aalborg, Denmark

Department of Autoimmunology and Biomarkers, Statens Serum Institut, DK-2300 Copenhagen, Denmark

Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark

⁴

Department of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus, Denmark

⁵

Department of Clinical Medicine, Aalborg University, DK-9000 Aalborg, Denmark

⁶

Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, University of Southern Denmark, DK-5000 Odense, Denmark

Authors to whom correspondence should be addressed.

^†

Contributed equally to this work.

^§

Deceased.

Biomedicines 2020, 8(8), 246; https://doi.org/10.3390/biomedicines8080246

Received: 22 June 2020 / Revised: 21 July 2020 / Accepted: 22 July 2020 / Published: 25 July 2020

(This article belongs to the Special Issue The Role of Extracellular Vesicles in Pathogenesis and Therapeutic Strategies)

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Abstract

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. View Full-Text

Keywords: Extracellular vesicles; human plasma; EV isolation; high-speed centrifugation; size exclusion chromatography; ME kit; peptide affinity; mass spectrometry; proteomics; proteome

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Askeland, A.; Borup, A.; Østergaard, O.; Olsen, J.V.; Lund, S.M.; Christiansen, G.; Kristensen, S.R.; Heegaard, N.H.H.; Pedersen, S. Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation. Biomedicines 2020, 8, 246. https://doi.org/10.3390/biomedicines8080246

AMA Style

Askeland A, Borup A, Østergaard O, Olsen JV, Lund SM, Christiansen G, Kristensen SR, Heegaard NHH, Pedersen S. Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation. Biomedicines. 2020; 8(8):246. https://doi.org/10.3390/biomedicines8080246

Chicago/Turabian Style

Askeland, Anders, Anne Borup, Ole Østergaard, Jesper V. Olsen, Sigrid M. Lund, Gunna Christiansen, Søren R. Kristensen, Niels H.H. Heegaard, and Shona Pedersen. 2020. "Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation" Biomedicines 8, no. 8: 246. https://doi.org/10.3390/biomedicines8080246

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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

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