Pooled healthy-person urine reveals urinary N-glycan diversity by reversed-phase LC/MS/MS

An analytical study of pooled urine samples from healthy individuals has produced an isomer-resolved dataset of human urinary N-glycans, documenting a broad range of molecular structures that included modified sugars, Lewis antigens, blood-group antigens, the Sda antigen and the HNK-1 antigen. The researchers combined reversed-phase liquid chromatography with tandem mass spectrometry in a workflow designed to distinguish glycan isomers and support automated structural assignment, creating a reference resource for future searches for low-abundance disease-associated urinary glycans.

Glycan microheterogeneity can sensitively reflect biological changes and has therefore been proposed as a promising source of biomarkers for early disease detection. Its analysis is complicated by high structural diversity, isomeric complexity and a wide dynamic range that separates abundant glycans from low-abundance species. The study addresses these analytical obstacles through an integrated platform built on pyridylamination, a chemical derivatization treatment that enables precise separation of glycan isomers by reversed-phase high-performance liquid chromatography.

The platform also incorporates retention behavior as a source of structural information. By quantifying the contribution of individual monosaccharide residues to retention, the researchers established a means of inferring unknown structures from the retention times of structurally related, known glycans. Normalized reversed-phase HPLC retention times were then integrated with detailed structural information obtained through chemical derivatization and MS/MS analysis. Together, these elements form a framework for isomer-discriminative analysis within a single reversed-phase LC/MS run and provide information that can be used for automated structural assignment.

Application of the strategy to pooled urine from healthy individuals comprehensively characterized urinary N-glycans carrying N-acetylneuraminic acid, sulfate, phosphate and glucuronic acid. The identified collection also included glycans bearing Lewis antigens, blood-group antigens, the Sda antigen and the HNK-1 antigen. Rather than limiting the dataset to simpler glycan compositions, the analysis resolves numerous structural variations and records how individual monosaccharide residues influence chromatographic retention. The resulting catalogue offers a foundation for detecting low-abundance, disease-associated urinary glycans and may help accelerate glycan biomarker discovery, although its reported application was to pooled healthy-person samples rather than a clinical performance evaluation.

Subject of Research: Isomer-resolved analysis of human urinary N-glycans for biomarker discovery

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

Article References: Makino, R., & Natsuka, S. (2026). Structural diversity of human urinary N-glycans revealed by reversed-phase LC/MS/MS toward a resource for biomarker discovery. Scientific Reports. https://doi.org/10.1038/s41598-026-77248-4

Image Credits: AI Generated

DOI: 10.1038/s41598-026-77248-4

Keywords: urinary N-glycans, glycomics, biomarker discovery, reversed-phase LC/MS/MS, glycan isomers, pyridylamination, tandem mass spectrometry, structural assignment, urine analysis, Structural, diversity, human