Glycoblotting enables seamless and straightforward workflow for MALDI-TOF/MS-based sulphoglycomics of N- and O-glycans.

Sulfated N- and O-glycans exist in trace levels which are challenging to detect, especially when abundant neutral and sialylated glycans are present. Current matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based sulfoglycomics approaches effectively utilize permethylation to discriminate sulfated glycans from sialyl-glycans. And a charge-based separation to isolate the sulfated glycans from the rest of the permethylated neutral and sialyl-glycans. However, these approaches suffer from concomitant sample losses during cleanup steps. Herein, we describe Glycoblotting as a straightforward complementary method with seamless glycan purification, enrichment, methylation, and labeling on a single platform to address sulfated glycan enrichment, sialic acid methylation, and sample loss. Glycoblottings' on-bead chemoselective ligation of reducing sugars with hydrazide showed excellent recovery of sulfated glycans, allowing the detection of more sulfated glycan species. On-bead methyl esterification of sialic acid using 3-methyl-1-p-tolyltriazene (MTT) effectively discriminates sulfated glycans from sialyl-glycans. Furthermore, we have shown that using MTT as a methylating agent allowed us to simultaneously detect and differentiate sulfate from phosphate groups in isobaric N-glycan species. We believe that Glycoblotting will contribute significantly to the MALDI-TOF MS-based Sulphoglycomics workflow.

Glycoblotting-Based Ovo-Sulphoglycomics Reveals Phosphorylated N-Glycans as a Possible Host Factor of AIV Prevalence in Waterfowls.

Sulfated N-glycans play a crucial role in the interaction between influenza A virus (IAV) and its host. These glycans have been found to enhance viral replication, highlighting their significance in IAV propagation. This study investigated the expression of acidic N-glycans, specifically sulfated and phosphorylated glycans, in the egg whites of 72 avian species belonging to the Order Anseriformes (waterfowls). We used the glycoblotting-based sulphoglycomics approach to elucidate the diversity of acidic N-glycans and infer their potential role in protecting embryos from infections. Family-specific variations in sulfated and phosphorylated N-glycan profiles were identified in waterfowl egg whites. Different waterfowl species exhibited distinct expressions of sulfated trans-Gal(+) and trans-Gal(-) N-glycan structures. Additionally, species-specific expression of phosphorylated N-glycans was observed. Furthermore, it was found that waterfowl species with high avian influenza virus (AIV) prevalence displayed a higher abundance of phosphorylated hybrid and high-mannose N-glycans on their egg whites. These findings shed light on the importance of phosphorylated and sulfated N-glycans in understanding the role of acidic glycans in IAV propagation.