Sialic acid methylation refines capillary electrophoresis laser-induced fluorescence analyses of immunoglobulin G N-glycans of ovarian cancer patients

Alterations in IgG N-glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE-LIF of 8-aminopyrene-1,3,6-trisulfonic acid labeled N-glycans is a well-established rapid method to characterize IgG N-glycans that needs only low amounts of starting material. However, sialylated N-glycans have short migration times due to their negative charge. As a result, some of them are not well resolved and co-migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgG N-glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co-migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented, which allowed an accurate quantification of these N-glycans. Finally, we investigated the IgG N-glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods. With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.