Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses
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 s...
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doaj-6ddb96dc48e0405ea2ade86b9765024f2020-11-25T00:23:33ZengTaylor & Francis GroupJournal of Extracellular Vesicles2001-30782017-12-016110.1080/20013078.2017.13594781359478Effects of storage temperature on airway exosome integrity for diagnostic and functional analysesRosario Maroto0Yingxin Zhao1Mohammad Jamaluddin2Vsevolod L. Popov3Hongwang Wang4Madumali Kalubowilage5Yueqing Zhang6Jonathan Luisi7Hong Sun8Christopher T. Culbertson9Stefan H. Bossmann10Massoud Motamedi11Allan R. Brasier12University of Texas Medical Branch (UTMB)University of Texas Medical Branch (UTMB)UTMBUTMBKansas State UniversityKansas State UniversityUTMBUTMBUTMBKansas State UniversityKansas State UniversityUTMBUniversity of Texas Medical Branch (UTMB)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.http://dx.doi.org/10.1080/20013078.2017.1359478Extracellular vesiclesbronchoalveolar lavagestorage conditionslabel-freequantitative proteomics |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Rosario Maroto Yingxin Zhao Mohammad Jamaluddin Vsevolod L. Popov Hongwang Wang Madumali Kalubowilage Yueqing Zhang Jonathan Luisi Hong Sun Christopher T. Culbertson Stefan H. Bossmann Massoud Motamedi Allan R. Brasier |
spellingShingle |
Rosario Maroto Yingxin Zhao Mohammad Jamaluddin Vsevolod L. Popov Hongwang Wang Madumali Kalubowilage Yueqing Zhang Jonathan Luisi Hong Sun Christopher T. Culbertson Stefan H. Bossmann Massoud Motamedi Allan R. Brasier Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses Journal of Extracellular Vesicles Extracellular vesicles bronchoalveolar lavage storage conditions label-free quantitative proteomics |
author_facet |
Rosario Maroto Yingxin Zhao Mohammad Jamaluddin Vsevolod L. Popov Hongwang Wang Madumali Kalubowilage Yueqing Zhang Jonathan Luisi Hong Sun Christopher T. Culbertson Stefan H. Bossmann Massoud Motamedi Allan R. Brasier |
author_sort |
Rosario Maroto |
title |
Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses |
title_short |
Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses |
title_full |
Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses |
title_fullStr |
Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses |
title_full_unstemmed |
Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses |
title_sort |
effects of storage temperature on airway exosome integrity for diagnostic and functional analyses |
publisher |
Taylor & Francis Group |
series |
Journal of Extracellular Vesicles |
issn |
2001-3078 |
publishDate |
2017-12-01 |
description |
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. |
topic |
Extracellular vesicles bronchoalveolar lavage storage conditions label-free quantitative proteomics |
url |
http://dx.doi.org/10.1080/20013078.2017.1359478 |
work_keys_str_mv |
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