Structural diversity of human urinary N-glycans revealed by reversed-phase LC/MS/MS toward a resource for biomarker discovery

This is the authors' abstract. We don't add key points for this paper.

Glycan microheterogeneity sensitively reflects biological changes and represents a promising source of biomarkers for early disease detection. However, the high structural diversity, isomeric complexity, and wide dynamic range of glycans remain major obstacles to comprehensive glycomic analysis, particularly for low-abundance species. To overcome these limitations, we developed an isomer-resolved N -glycan dataset that supports automated structural assignment. The platform is based on pyridylamination, which enables precise separation of glycan isomers according to reversed-phase HPLC. Furthermore, by quantifying the contribution of individual monosaccharide residues to retention behavior, unknown structures can be inferred from the retention times of structurally related known glycans. By integrating normalized reversed-phase HPLC retention times with detailed structural information obtained through chemical derivatization and MS/MS analysis, we established a framework for accurate isomer-discriminative analysis in a single reversed-phase LC/MS run. We applied this strategy to pooled urine samples of healthy individuals and comprehensively characterized urinary N -glycans, including structures carrying N -acetylneuraminic acid, sulfate group, phosphate group, glucuronic acid, Lewis antigens, blood group antigens, Sda antigen, and HNK-1 antigen. This dataset provides a foundation for detecting low-abundance disease-associated urinary glycans and may accelerate glycan biomarker discovery.