Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses

Rosario Maroto, Yingxin Zhao, Mohammad Jamaluddin, Vsevolod Popov, Hongwang Wang, Madumali Kalubowilage, Yueqing Zhang, Jonathan Luisi, Hong Sun, Christopher T. Culbertson, Stefan H. Bossmann, Massoud Motamedi, Allan R. Brasier

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

Original languageEnglish (US)
Article number1359478
JournalJournal of Extracellular Vesicles
Volume6
Issue number1
DOIs
StatePublished - Dec 1 2017

Fingerprint

Exosomes
Temperature
Proteins
Bronchoalveolar Lavage Fluid
Biological Phenomena
Surface Properties
Ultracentrifugation
Transmission Electron Microscopy

Keywords

  • bronchoalveolar lavage
  • Extracellular vesicles
  • label-free
  • quantitative proteomics
  • storage conditions

ASJC Scopus subject areas

  • Histology
  • Cell Biology

Cite this

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses. / Maroto, Rosario; Zhao, Yingxin; Jamaluddin, Mohammad; Popov, Vsevolod; Wang, Hongwang; Kalubowilage, Madumali; Zhang, Yueqing; Luisi, Jonathan; Sun, Hong; Culbertson, Christopher T.; Bossmann, Stefan H.; Motamedi, Massoud; Brasier, Allan R.

In: Journal of Extracellular Vesicles, Vol. 6, No. 1, 1359478, 01.12.2017.

Research output: Contribution to journalArticle

Maroto, R, Zhao, Y, Jamaluddin, M, Popov, V, Wang, H, Kalubowilage, M, Zhang, Y, Luisi, J, Sun, H, Culbertson, CT, Bossmann, SH, Motamedi, M & Brasier, AR 2017, 'Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses', Journal of Extracellular Vesicles, vol. 6, no. 1, 1359478. https://doi.org/10.1080/20013078.2017.1359478
Maroto, Rosario ; Zhao, Yingxin ; Jamaluddin, Mohammad ; Popov, Vsevolod ; Wang, Hongwang ; Kalubowilage, Madumali ; Zhang, Yueqing ; Luisi, Jonathan ; Sun, Hong ; Culbertson, Christopher T. ; Bossmann, Stefan H. ; Motamedi, Massoud ; Brasier, Allan R. / Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses. In: Journal of Extracellular Vesicles. 2017 ; Vol. 6, No. 1.
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abstract = "Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10{\%} increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25{\%} increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.",
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author = "Rosario Maroto and Yingxin Zhao and Mohammad Jamaluddin and Vsevolod Popov and Hongwang Wang and Madumali Kalubowilage and Yueqing Zhang and Jonathan Luisi and Hong Sun and Culbertson, {Christopher T.} and Bossmann, {Stefan H.} and Massoud Motamedi and Brasier, {Allan R.}",
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AU - Maroto, Rosario

AU - Zhao, Yingxin

AU - Jamaluddin, Mohammad

AU - Popov, Vsevolod

AU - Wang, Hongwang

AU - Kalubowilage, Madumali

AU - Zhang, Yueqing

AU - Luisi, Jonathan

AU - Sun, Hong

AU - Culbertson, Christopher T.

AU - Bossmann, Stefan H.

AU - Motamedi, Massoud

AU - Brasier, Allan R.

PY - 2017/12/1

Y1 - 2017/12/1

N2 - Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

AB - Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

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KW - Extracellular vesicles

KW - label-free

KW - quantitative proteomics

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