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Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for Ap...

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Autores principales: Ter-Ovanesyan, Dmitry, Gilboa, Tal, Budnik, Bogdan, Nikitina, Adele, Whiteman, Sara, Lazarovits, Roey, Trieu, Wendy, Kalish, David, Church, George M, Walt, David R
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10229111/
https://www.ncbi.nlm.nih.gov/pubmed/37252755
http://dx.doi.org/10.7554/eLife.86394
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author Ter-Ovanesyan, Dmitry
Gilboa, Tal
Budnik, Bogdan
Nikitina, Adele
Whiteman, Sara
Lazarovits, Roey
Trieu, Wendy
Kalish, David
Church, George M
Walt, David R
author_facet Ter-Ovanesyan, Dmitry
Gilboa, Tal
Budnik, Bogdan
Nikitina, Adele
Whiteman, Sara
Lazarovits, Roey
Trieu, Wendy
Kalish, David
Church, George M
Walt, David R
author_sort Ter-Ovanesyan, Dmitry
collection PubMed
description Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.
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spelling pubmed-102291112023-05-31 Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins Ter-Ovanesyan, Dmitry Gilboa, Tal Budnik, Bogdan Nikitina, Adele Whiteman, Sara Lazarovits, Roey Trieu, Wendy Kalish, David Church, George M Walt, David R eLife Biochemistry and Chemical Biology Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery. eLife Sciences Publications, Ltd 2023-05-30 /pmc/articles/PMC10229111/ /pubmed/37252755 http://dx.doi.org/10.7554/eLife.86394 Text en © 2023, Ter-Ovanesyan, Gilboa et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Ter-Ovanesyan, Dmitry
Gilboa, Tal
Budnik, Bogdan
Nikitina, Adele
Whiteman, Sara
Lazarovits, Roey
Trieu, Wendy
Kalish, David
Church, George M
Walt, David R
Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
title Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
title_full Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
title_fullStr Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
title_full_unstemmed Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
title_short Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
title_sort improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10229111/
https://www.ncbi.nlm.nih.gov/pubmed/37252755
http://dx.doi.org/10.7554/eLife.86394
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