The secreted cellular sub-
proteome (secretome) 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-overexpressing subtype (SKBR3), and basal B subtype (MDA-MB157, MDA-MB231, HS578T). On the basis of intact molecular mass, LC retention time, and MS/MS fragmentation, 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 paucimannose and complex type N-
glycans containing bisecting β1,4-GlcNAc and
LacdiNAc determinants. Subtype-specific glycosylation were observed, including the preferential expression of α2,3-sialylation in the basal B
breast cancer cells. Pathway analysis indicated that the regulated N-
glycans were biosynthetically related. Tight clustering of the
breast cancer subtypes based on N-glycome signatures supported the involvement of N-glycosylation in
cancer. In conclusion, we are the first to report on the secretome N-glycosylation of a panel of breast epithelial cell lines representing different subtypes. Complementing
proteome and
lipid profiling, N-glycome mapping yields important pieces of structural information to help understand the biomolecular deregulation in
breast cancer development and progression, knowledge that may facilitate the discovery of candidate
cancer markers and potential
drug targets.