Comprehensive N-glycome profiling of cultured human epithelial breast cells identifies unique secretome N-glycosylation signatures enabling tumorigenic sub-type classification

The secreted cellular sub-proteome is a rich source of biologically active glycoproteins. N-glycan profiling of secretomes of cultured cancer cells provides an opportunity to investigate the link between protein N-glycosylation and tumorigenesis. Utilizing carbon-LC-ESI-CID-MS/MS of protein released native N-glycans, we accurately profiled the secretome N-glycosylation of six human epithelial breast cells including normal mammary epithelial cells (HMEC) and breast cancer cells belonging to luminal A subtype (MCF7), HER2-overexpressed subtype (SKBR3) and basal B subtype (MDA-MB157, MDA-MB231, HS578T). Based on intact molecular mass, LC retention time and fragmentation pattern, a total of 74 N-glycans were confidently identified and quantified. The secretomes comprised significant levels of highly sialylated and fucosylated complex type N-glycans, which were elevated in all cancer cells relative to HMEC (57.7-87.2% vs 24.9%, p < 0.0001 and 57.1-78.0% vs 38.4%, p < 0.0001-0.001 respectively). Similarly, other glycan features were found to be altered in breast cancer secretomes including expression of paucimannose N-glycans, complex type N-glycans containing bisecting β1,4-GlcNAc and LacdiNAc determinants. Subtype-specific glycosylation patterns were observed. Prominently, the basal B breast cancer cells expressed preferentially N-glycan α2,3-sialylation. Pathway analysis indicated that the regulated N-glycans were closely related in the biosynthetic machinery. Tight clustering of the breast cancer sub-types based on the obtained N-glycome profiles supported the involvement of N-glycosylation in cancer. Complementing proteome and lipid profiling, N-glycome mapping yields important pieces of structural information to understand the biomolecular de-regulation in breast cancer development and progression - knowledge which may facilitate the discovery of candidate cancer markers and potential drug targets.