Concerted mass spectrometry-based glycomic approach for precision mapping of sulfo sialylated N-glycans on human peripheral blood mononuclear cells and lymphocytes.

Despite well-recognized biological importance, mass spectrometry (MS)-based glycomic identification of sulfo-, sialylated terminal glyco-epitopes on the N-glycans of various immune cell types remains technically challenging and rarely reported. Previous studies with monoclonal antibody have implicated a regulated expression of 6-sulfo-α2-6-sialyl LacNAc on B cells in peripheral lymph nodes and the circulating peripheral blood lymphocytes but its occurrence on leukemia cells or lymphomas have not been critically addressed. In this study, we have extended our previously developed MS-based sulfoglycomic platform by incorporating additional complementary analytical approaches in order to achieve a high sensitivity mapping and relative quantification of the detected sulfated glycotopes down to the level of defining their sialyl linkages. We showed that discovery mode sulfoglycomics and precise location of sulfate were best achieved by multimode MS analyses of fractionated, permethylated sulfated N-glycans. On the other hand, the relative degree of sulfation on individual N-glycans could be more efficiently inferred from the respective extracted ion chromatograms of native, non-sulfated and sulfated target N-glycans in single LC-MS/MS runs. The GlcNAc-6-O-sulfated α2-6-sialyl LacNAc, which constitutes the higher affinity ligand for the human inhibitory co-receptor of B cells, CD22, was found to be commonly carried on a range of complex type N-glycans from human CD19+ and CD4+ lymphocytes. We further showed that its occurrence on the most abundant α2-6-disialylated biantennary structure from the peripheral blood mononuclear cells of patients diagnosed as B-cell chronic lymphocytic leukemia varied within ±2-fold abundance from the mean value determined for isolated CD19+ lymphocytes and cultured B-CLL cells.