Navigating the Landscape of Native O-glycomic Analysis Across Diverse Biological Systems

Glycosylation, a post translations modification, plays a central role in the structure and function of nearly all membrane and secreted proteins. It is categorized into two types: N-linked and O-linked glycans, with N-glycan analysis widely established through enzymatic release. The heterogeneity of the O-glycans, stemming from the lack of a common glycan core for enzyme cleavage, presents additional challenges to the analysis. Despite its complexity, the study of O-glycans remains crucial in identifying O-glycan biomarkers for glyco-therapeutics and investigating various diseases. We have developed a chemical method optimized for mass spectrometry (MS) analysis that allows us to liberate and identify O-glycans in a variety of different biological samples spanning from vaginal swabs to organ-on-chip mucus to brain tissue. In women affected by BV, O-glycomic analysis revealed the weakening of the mucus layer in the vaginal cavity evident through the decrease in sialylated structures indicating a disruption in the protective barrier. These findings were supported with vaginal organ-on-a-chip systems demonstrating similar behavior upon exposure to the BV bacteria consortia. In brain tissue the O-glycomic mapping revealed differences in the O-glycoproteins expressed between healthy individuals and patients diagnosed with Alzheimer's disease (AD) at various stages. This developed analytical workflow can easily be adapted to samples of any nature to approach various biological inquires.