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

Abstract

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.

Multiple Roles of Glycans in Hematological Malignancies

“…Most discoveries of aberrant glycosylation in hematological malignancy are based on genomics analysis using patient samples, mostly focusing on the abnormal expression of genes for glycosyltransferases and glycosidases. In recent years, the development of mass spectrometry-based proteomic and glycomic techniques make it possible to discover the abnormal glycoproteins and glycolipids, and cancer-related and glycosite-specific glycan structures, with the limited samples ( 144 ). Tracing back the abnormal glycan structures to the corresponding genes may sparkle this field in hematological malignancy study.…”

Systems glycomics of adult zebrafish identifies organ-specific sialylation and glycosylation patterns

“…Cartridge was washed with methanol/water (1:1, v/v) and eluted successively by methanol, chloroform/methanol (1:1, v/v) and (2:1, v/v). For the purification of N-glycans from brain regions, an alternative methodology based on the extraction of glycocoproteins by Triton X-100 has been used 69 . In brief, tissues were suspended in 200 μL of 1% triton in PBS and sonicated three times for 10 s. Cell debris were removed by centrifugation at 10,000× g , 4 °C for 10 min and a final concentration of 10 mM dithiothreitol and 50 mM iodoacetamide in 100 μL PBS were added sequentially to the supernatant, each followed by incubation at 37 °C for 1 h. Then 44 μL of 100% trichloroacetic acid was added and left at −20 °C for 30 min.…”

Novel lamprey antibody recognizes terminal sulfated galactose epitopes on mammalian glycoproteins

“…The presence of 3-O-SGal is regulated by Gal3ST-2 and Gal3ST-3, both which can sulfate type-2 LacNAc (Galβ1-4GlcNAc) 21 on N-glycans, whereas Gal3ST-4 prefers Galβ1-3GalNAc on O-glycans 21 , 22 . Despite its importance as a terminal glycan modification, 3-O-SGal has been difficult to study and analyze because it is relatively labile and analysis commonly require special procedural modifications 23 – 26 . There are no reagents for directly exploring 3-O-SGal expression, thus its frequency and distribution in mammalian tissues and cells are poorly described.…”