iProXProteomicsChunfang GaoHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0010968000gaocf1115@163.comShanghai University of Traditional Chinese Medicine, Yueyang Hospital of Integrated Traditional Chinese and Western MedicineiProX<h4>Background</h4>Extracellular vesicles (EVs) are nanoscale structures involved in intercellular communication and play a key role in cancer pathology. Intrahepatic cholangiocarcinoma (ICC) is a highly invasive malignancy marked by abnormal sialylated glycosylation. Analyzing proteins and glycans in EVs provides insights into ICC molecular subtyping and mechanisms. Optimizing EV isolation methods for ICC-derived EVs enables comprehensive proteomic and glycomic analysis.<h4>Methods</h4>We systematically evaluated five EV isolation methods-Ultracentrifugation (UC), exoEasy, Total Exosome Isolation (TEI), EVtrap, and ÄKTA-by analyzing the biophysical properties, proteomic profiles, and glycomic structures of EVs. Subsequently, we applied TMT-based quantitative proteome and light/heavy methylamine labeling for the quantification of sialylated N-glycan linkage isomers to investigate alterations in proteins and N-glycans within EVs secreted by HuCCT1 and HCCC-9810 cells with overexpressing ST6 β‑galactoside α2,6‑sialyltransferase 1 (ST6GAL1).<h4>Results</h4>By evaluating the biophysical properties, proteome, and N-glycome of EVs extracted using five different methods, UC was identified as the optimal approach for this study, as it offered a balance between operational complexity, cost-effectiveness, and the preservation of EVs activity. In this study, a total of 1,928 high-confidence proteins and over 84 high-confidence glycans were quantified. EVs secreted by HuCCT1 and HCCC-9810 cells overexpressing ST6GAL1 exhibited consistent upregulation of 16 proteins, consistent downregulation of 10 proteins, as well as consistent upregulation of 3 glycans and consistent downregulation of 3 glycans.<h4>Conclusions</h4>Quantitative proteomic and glycomic analysis of ICC-derived EVs revealed that ST6GAL1 overexpression led to significant alterations in proteins involved in cancer cell adhesion and glycosylation pathways, along with specific changes in N-glycan structures. Notably, these modifications extended beyond α2,6-sialylation, suggesting that interactions between glycosyltransferases and glycans may drive these alterations.Comparative evaluation of methods for isolating extracellular vesicles from ICC cell culture supernatants: Insights into proteomic and glycomic analysis.Wu Linlin L, Wei Jiao J, Zhan Yueping Y, Xiao Xiao X, Xu Xuewen X, Huang Chenjun C, Long Tian T, Li Yueyue Y, Fu Bin B, Wang Mengmeng M, Gao Chunfang CfalseComparative evaluation of methods for isolating extracellular vesicles from ICC cell culture supernatants: Insights into proteomic and glycomic analysisExtracellular vesicles (EVs) are specialized nanoscale vesicular structures that act as mediators of intercellular communication, playing crucial roles in the pathological processes of cancers. Aberrant glycosylation is a hallmark of cancer, the analysis of EVs and protein glycosylation provides valuable insights into cancer research. Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive and insidious malignancy, characterized by significant abnormalities in sialylation glycosylation. This study focused on the comprehensive analysis of the biophysical properties and cargo of EVs secreted by ICC cells. First, we evaluated five EVs isolation methods and identified ultracentrifugation (UC) as the optimal technique. Subsequently, we applied TMT-based quantitative proteome and light/heavy methylamine labeling for the quantification of sialylated N-glycan linkage isomers to investigate alterations in proteins and N-glycans within EVs secreted by HuCCT1 and HCCC-9810 cells overexpressing ST6GAL1. This integrated analytical strategy enhanced our ability to quantify EVs proteome and N-glycome in conditioned media, allowing us to quantify over 2,292 proteins and 65 N-glycans in ICC-derived EVs. The results revealed that ST6GAL1 overexpression induced variations in proteins associated with cancer cell adhesion and glycosylation pathways, as well as alterations in specific N-glycan structures. In addition, our comprehensive evaluation also considered the bioactivity of the extracted EVs, making them suitable for subsequent bioanalytical studies. In the future, we will focus on mechanistic investigations to elucidate the impact of ST6GAL1-induced EVs protein glycosylation changes on the pathogenesis of ICC.Mon Mar 24 00:00:00 GMT 2025PXD062160960640301937