LC-MS/MS analysis of surface and lysate N-glycans of CHO-K1 cells: Structure, relative quantity, and absolute quantity

Chinese hamster ovary (CHO)-K1 cells are widely used in biomedical research relevant to cancer, toxicity screening, and viruses, as well as in the production of recombinant proteins for biopharmaceuticals. In this study, liquid chromatography (LC)-electrospray ionization (ESI)-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS) was used to characterize the surface and lysate N-glycans of CHO-K1 cells and analyze their structures. The relative quantity (%) of each N-glycan and absolute quantity (pmol) of total N-glycans were also obtained. In total, 37 surface and 30 lysate N-glycans were identified. Each of these two fractions contained eight high-mannose type (required for protection against proteolysis and N-glycosylation of recombinant proteins) at 28.8 % (the sum of the relative quantities of each N-glycan) and 66.5 %, respectively. Additionally, the surface and lysate N-glycans differed in their levels of sialyation (affect cell-cell interactions; 48.1 % and 13.5 %), fucosylation (affect cell signaling; 37.9 % and 25.5 %), and terminal-galactosylation (prerequisite for subsequent sialylation; 36.6 % and 20.9 %). These results indicate that the lysate of CHO-K1 cells contained more mannosylated (2.3-fold) N-glycans compared to the surface, which contained relatively more sialylated (3.6-fold), slightly more highly fucosylated (1.5-fold), and more terminal-galactosylated (1.8-fold) N-glycans. The sum of the absolute quantity of each N-glycan was obtained as a ratio of 1 (1,778.7 pmol; surface):2.2 (3,887.3 pmol; lysate) from approximately 5 × 10⁶ CHO-K1 cells. This study is the first to compare the surface and lysate N-glycans of CHO-K1 cells using LC-ESI-HCD-MS/MS. The results can be used to control and optimize biotechnology and biomedical research using CHO-K1 cells.