Comparative evaluation of methods for isolating extracellular vesicles from ICC cell culture supernatants: Insights into proteomic and glycomic analysis DOI Creative Commons
Linlin Wu, Jiao Wei, Yueping Zhan

et al.

Cell Communication and Signaling, Journal Year: 2025, Volume and Issue: 23(1)

Published: April 29, 2025

Extracellular vesicles (EVs) are nanoscale structures involved in intercellular communication and play a key role cancer pathology. Intrahepatic cholangiocarcinoma (ICC) is highly invasive malignancy marked by abnormal sialylated glycosylation. Analyzing proteins glycans EVs provides insights into ICC molecular subtyping mechanisms. Optimizing EV isolation methods for ICC-derived enables comprehensive proteomic glycomic analysis. We systematically evaluated five methods-Ultracentrifugation (UC), exoEasy, Total Exosome Isolation (TEI), EVtrap, ÄKTA-by analyzing the biophysical properties, profiles, of EVs. Subsequently, we applied TMT-based quantitative proteome light/heavy methylamine labeling quantification N-glycan linkage isomers to investigate alterations N-glycans within secreted HuCCT1 HCCC-9810 cells with overexpressing ST6 β‑galactoside α2,6‑sialyltransferase 1 (ST6GAL1). By evaluating proteome, N-glycome extracted using different methods, UC was identified as optimal approach this study, it offered balance between operational complexity, cost-effectiveness, preservation activity. In total 1,928 high-confidence over 84 were quantified. ST6GAL1 exhibited consistent upregulation 16 proteins, downregulation 10 well 3 glycans. Quantitative analysis revealed that overexpression led significant cell adhesion glycosylation pathways, along specific changes structures. Notably, these modifications extended beyond α2,6-sialylation, suggesting interactions glycosyltransferases may drive alterations.

Language: Английский

Comparative evaluation of methods for isolating extracellular vesicles from ICC cell culture supernatants: Insights into proteomic and glycomic analysis DOI Creative Commons
Linlin Wu, Jiao Wei, Yueping Zhan

et al.

Cell Communication and Signaling, Journal Year: 2025, Volume and Issue: 23(1)

Published: April 29, 2025

Extracellular vesicles (EVs) are nanoscale structures involved in intercellular communication and play a key role cancer pathology. Intrahepatic cholangiocarcinoma (ICC) is highly invasive malignancy marked by abnormal sialylated glycosylation. Analyzing proteins glycans EVs provides insights into ICC molecular subtyping mechanisms. Optimizing EV isolation methods for ICC-derived enables comprehensive proteomic glycomic analysis. We systematically evaluated five methods-Ultracentrifugation (UC), exoEasy, Total Exosome Isolation (TEI), EVtrap, ÄKTA-by analyzing the biophysical properties, profiles, of EVs. Subsequently, we applied TMT-based quantitative proteome light/heavy methylamine labeling quantification N-glycan linkage isomers to investigate alterations N-glycans within secreted HuCCT1 HCCC-9810 cells with overexpressing ST6 β‑galactoside α2,6‑sialyltransferase 1 (ST6GAL1). By evaluating proteome, N-glycome extracted using different methods, UC was identified as optimal approach this study, it offered balance between operational complexity, cost-effectiveness, preservation activity. In total 1,928 high-confidence over 84 were quantified. ST6GAL1 exhibited consistent upregulation 16 proteins, downregulation 10 well 3 glycans. Quantitative analysis revealed that overexpression led significant cell adhesion glycosylation pathways, along specific changes structures. Notably, these modifications extended beyond α2,6-sialylation, suggesting interactions glycosyltransferases may drive alterations.

Language: Английский

Citations

0