Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study

Tímea Böröczky; Gabriella Dobra; Mátyás Bukva; Edina Gyukity-Sebestyén; Éva Hunyadi-Gulyás; Zsuzsanna Darula; Péter Horváth; Krisztina Buzás; Mária Harmati

doi:10.3390/life13010206

Life (Jan 2023)

Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study

Tímea Böröczky,
Gabriella Dobra,
Mátyás Bukva,
Edina Gyukity-Sebestyén,
Éva Hunyadi-Gulyás,
Zsuzsanna Darula,
Péter Horváth,
Krisztina Buzás,
Mária Harmati

Affiliations

Tímea Böröczky: Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Gabriella Dobra: Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Mátyás Bukva: Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Edina Gyukity-Sebestyén: Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Éva Hunyadi-Gulyás: Laboratory of Proteomics Research, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Zsuzsanna Darula: Laboratory of Proteomics Research, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Péter Horváth: Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Krisztina Buzás: Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary
Mária Harmati: Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, Hungary

DOI: https://doi.org/10.3390/life13010206
Journal volume & issue: Vol. 13, no. 1
p. 206

Abstract

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Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the key role of EVs in intercellular communication and pathophysiological processes. However, the heterogeneity of EVs challenges their exploration and the establishment of gold-standard methods. Here, we aimed to reveal the influence of technical changes on EV biology and the reliability of experimental data. We used B16F1 melanoma cells as a model and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway enrichment analysis to analyze the quantity, size distribution, proteome and function of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing medium or serum-free medium. Additionally, we investigated the effects of minor technical variances on the quality of sEV preparations. We found that storage of the isolates at −80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step after differential ultracentrifugation has a minor influence on the sEV proteome. In contrast, FBS starvation affects the production and proteome of sEVs; moreover, these vesicles may have a greater impact on protein metabolism, but a smaller impact on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated that FBS starvation has a strong influence on sEV biology, applying serum-free conditions might be considered in in vitro sEV studies.

Published in Life

ISSN: 2075-1729 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science
Website: http://www.mdpi.com/journal/life

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