Cell-surface N-glycans play important roles in both inter- and intracellular processes, including cell adhesion and development, cell recognition, as well as cancer development and metastasis; detailed structural characterization of these N-glycans is thus paramount. Here we report our comparative N-glycomics study of cell-surface N-glycans of the hepatocellular carcinoma (HCC) HepG2 cells vs the normal liver LO2 cells. With sequential trypsin digestion of proteins, C18 depletion of peptides without glycosylation, PNGase F digestion of N-glycopeptides, PGC enrichment of N-glycans, CH₃I permethylation of the enriched N-glycans, cell-surface N-glycomes of the HepG2 and LO2 cells were analyzed using C18-RPLC–MS/MS (HCD). With spectrum-level FDR no bigger than 1%, 351 and 310 N-glycans were identified for HepG2 and LO2, respectively, with comprehensive structural information (not only monosaccharide composition, but also sequence and linkage) by N-glycan database search engine GlySeeker. The percentage of hybrid N-glycans with tetra-antennary structures was substantially increased in the HepG2 cells. This comprehensive discovery study of differentially expressed cell-surface N-glycans in HepG2 vs LO2 serves as a solid reference for future validation study of glycosylation markers in HCC.

中文翻译：

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HepG2与LO2细胞系的细胞表面N-糖苷的比较糖蛋白研究

细胞表面N-聚糖在细胞间和细胞内过程中都起着重要作用，包括细胞粘附和发育，细胞识别以及癌症的发生和转移。因此，这些N-聚糖的详细结构表征至关重要。在这里，我们报告了肝细胞癌（HCC）HepG2细胞与正常肝LO2细胞的细胞表面N-聚糖的比较N糖组学研究。依次进行胰蛋白酶消化蛋白质，不进行糖基化的肽的C18消耗，N-糖肽的PNGase F消化，N-糖的PGC富集，CH ₃使用C18-RPLC-MS / MS（HCD）分析了富集的N聚糖的I甲基化，HepG2和LO2细胞的细胞表面N糖基。在谱级FDR不大于1％的情况下，N-聚糖数据库搜索引擎GlySeeker分别为HepG2和LO2鉴定了351和310个N-聚糖，并提供了全面的结构信息（不仅是单糖组成，还包括序列和链接）。 。在HepG2细胞中，具有四天线结构的杂合N-聚糖的百分比显着增加。这项在HepG2与LO2中差异表达的细胞表面N-聚糖的全面发现研究为将来在HCC中糖基化标记的验证研究提供了坚实的参考。