Sulfoglycomic identification of sulfated glycotopes underlies subsequent development of target-driven glycoproteomic workflows

Glycans carried on proteins in rodent and human brains are well characterized by the expression of terminal GlcNAc, LacNAc, sialyl-LacNAc, LeX, and a low level of disialylated LacNAc in two different settings. In contrast, despite the well-known presence of the HNK-1 epitope, the sulfated glycotopes have rarely been analyzed and reported in present-day glycomic efforts, presumably due to their low abundance and unfavorable ionization properties for efficient mass spectrometry detection. Similarly, none of the many reported brain N-glycoproteomics to date have confidently identified any specific protein carriers of sulfated N-glycans, which severely impeded progress in gaining further glycobiology insights into how additional sulfation on glycans may modulate functions mediated by protein site-specific glycosylation. By our unique sulfoglycomic workflow based on negative mode MS analysis of permethylated glycans, we have, over the years, resolved and definitively identified a myriad of sulfated glycotopes carried on the mouse and human brains, including internal 6-sulfo-GlcNAc of a LacNAc, terminal 3 or/and 6-sulfo Gal, terminal 4-sulfo-GalNAc of a LacdiNAc, and HNK-1. Intriguingly, we have unambiguously shown that the core fucose of a subset of N-glycans, from simple truncated paucimannose to complex type multiantennary structures, could be sulfated, by a combination of diagnostic MS2 ions. Furthermore, glial and neuron cells isolated from murine cortex/striatum displayed cell type-specific expression of core Fuc-sulfation, whereby neurons carried the highest level of core Fuc-sulfated N-glycan structures, with lesser amount in astrocytes, and not detected in microglia. The regional and cell type-specific sulfoglycomic profiles are largely supported by the differential expression of the mRNA transcripts of various known and putative carbohydrate sulfotransferases. These could be manipulated to derive model cells and glycopeptides enriched with the target sulfated glycotopes, thereby serving as invaluable standards and models for our ongoing development of target-driven sulfo-glycoproteomic workflows.