Comparative Glycomics Study of Cell-Surface N‑Glycomes of HepG2 versus LO2 Cell Lines

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.