Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study
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 revea...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-01-01
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| Series: | Life |
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| Online Access: | https://www.mdpi.com/2075-1729/13/1/206 |
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| author | Tímea Böröczky Gabriella Dobra Mátyás Bukva Edina Gyukity-Sebestyén Éva Hunyadi-Gulyás Zsuzsanna Darula Péter Horváth Krisztina Buzás Mária Harmati |
| author_facet | Tímea Böröczky Gabriella Dobra Mátyás Bukva Edina Gyukity-Sebestyén Éva Hunyadi-Gulyás Zsuzsanna Darula Péter Horváth Krisztina Buzás Mária Harmati |
| author_sort | Tímea Böröczky |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T11:56:04Z |
| format | Article |
| id | doaj.art-ec42856c72394426b414851410edd57f |
| institution | Directory Open Access Journal |
| issn | 2075-1729 |
| language | English |
| last_indexed | 2024-03-09T11:56:04Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Life |
| spelling | doaj.art-ec42856c72394426b414851410edd57f2023-11-30T23:09:28ZengMDPI AGLife2075-17292023-01-0113120610.3390/life13010206Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative StudyTímea Böröczky0Gabriella Dobra1Mátyás Bukva2Edina Gyukity-Sebestyén3Éva Hunyadi-Gulyás4Zsuzsanna Darula5Péter Horváth6Krisztina Buzás7Mária Harmati8Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Proteomics Research, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Proteomics Research, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryLaboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eötvös Lorand Research Network (ELKH), H-6726 Szeged, HungaryExtracellular 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.https://www.mdpi.com/2075-1729/13/1/206small extracellular vesicles (sEVs)sEV proteomemass spectrometrynanoparticle tracking analysis (NTA)pathway enrichment analysisEV-depleted fetal bovine serum (FBS) |
| spellingShingle | Tímea Böröczky Gabriella Dobra Mátyás Bukva Edina Gyukity-Sebestyén Éva Hunyadi-Gulyás Zsuzsanna Darula Péter Horváth Krisztina Buzás Mária Harmati Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study Life small extracellular vesicles (sEVs) sEV proteome mass spectrometry nanoparticle tracking analysis (NTA) pathway enrichment analysis EV-depleted fetal bovine serum (FBS) |
| title | Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study |
| title_full | Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study |
| title_fullStr | Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study |
| title_full_unstemmed | Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study |
| title_short | Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study |
| title_sort | impact of experimental conditions on extracellular vesicles proteome a comparative study |
| topic | small extracellular vesicles (sEVs) sEV proteome mass spectrometry nanoparticle tracking analysis (NTA) pathway enrichment analysis EV-depleted fetal bovine serum (FBS) |
| url | https://www.mdpi.com/2075-1729/13/1/206 |
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