Glycans of immune cells and immunoglobulins play essential roles in their biological functions, however there is a lack of methods to induce targeted changes to these glycans and characterize the changes via MS based techniques. Here we propose a method that allows the modification and characterization of immune cell surface and the secreted antibody glycans. Metabolic engineering tools, such as monosaccharide supplementation and glycosyltransferase inhibition, were employed for selective modification of the glycans, and both immune cell surface glycans released from the cell membrane and secreted antibody glycans were analyzed through MS based glycomics and glycoproteomics analyses. Cell surface glycomics data supported the hypothesis that the immune cell glycans can be modified selectively using glycosyltransferase inhibitors. For example, treatment of the cells with a glycan sialylation inhibitor, 3-Fluorinated sialic acid, reduced the relative abundances of sialofucosylated and sialylated cell surface glycans by 25% and 3% respectively, and the relative abundances of fucosylated glycans were increased by 22%, indicating the effectiveness of this treatment in inhibition of sialylated and sialofucosylated glycan formation. Site specific glycoproteomics analysis of immunoglobulins secreted from the same cells showed the glycans at site Asn 297 of IgG underwent the same changes as the cell surface glycans. Glycans at Asn 297 of IgG play a vital role in functionality of these antibodies. The monosaccharide supplementations, such as fructose, resulted in diversification of the N-glycan structures at Asn 297 and identification of unique structures with one glycan containing 5 hexoses, 5 N-acetylhexosamines, 1 Fucose and 0 N-acetylneuraminic acid (sialic acid), which was not observed in the control samples. Therefore, the presence or absence of different additives in human lymphoblast cell matrix does not only affect the cell surface glycosylation, but also affects the glycosylation of the secreted antibodies from these cells.

Characterization of induced glycosylation changes of immune cells and antibodies

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