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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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.

I: Biomedicines, Bind 8, Nr. 8, 246, 2020.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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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. I: Biomedicines. 2020 ; Bind 8, Nr. 8.

Bibtex

@article{f8321898b7414e9c9b6019e860e0c32f,
title = "Mass-spectrometry based proteome comparison of extracellular vesicle isolation methods: Comparison of ME-kit, size-exclusion chromatography, and high-speed centrifugation",
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.",
keywords = "EV isolation, Extracellular vesicles, High-speed centrifugation, Human plasma, Mass spectrometry, ME kit, Peptide affinity, Proteome, Proteomics, Size exclusion chromatography",
author = "Anders Askeland and Anne Borup and Ole {\O}stergaard and Olsen, {Jesper V.} and Lund, {Sigrid M.} and Gunna Christiansen and Kristensen, {S{\o}ren R.} and Heegaard, {Niels H.H.} and Shona Pedersen",
year = "2020",
doi = "10.3390/BIOMEDICINES8080246",
language = "English",
volume = "8",
journal = "Biomedicines",
issn = "2227-9059",
publisher = "MDPI",
number = "8",

}

RIS

TY - JOUR

T1 - Mass-spectrometry based proteome comparison of extracellular vesicle isolation methods

T2 - Comparison of ME-kit, size-exclusion chromatography, and high-speed centrifugation

AU - Askeland, Anders

AU - Borup, Anne

AU - Østergaard, Ole

AU - Olsen, Jesper V.

AU - Lund, Sigrid M.

AU - Christiansen, Gunna

AU - Kristensen, Søren R.

AU - Heegaard, Niels H.H.

AU - Pedersen, Shona

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - EV isolation

KW - Extracellular vesicles

KW - High-speed centrifugation

KW - Human plasma

KW - Mass spectrometry

KW - ME kit

KW - Peptide affinity

KW - Proteome

KW - Proteomics

KW - Size exclusion chromatography

UR - http://www.scopus.com/inward/record.url?scp=85089669803&partnerID=8YFLogxK

U2 - 10.3390/BIOMEDICINES8080246

DO - 10.3390/BIOMEDICINES8080246

M3 - Journal article

AN - SCOPUS:85089669803

VL - 8

JO - Biomedicines

JF - Biomedicines

SN - 2227-9059

IS - 8

M1 - 246

ER -