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
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.
