Nanopore profiling and structure determination of oligosaccharides by fragmentation
2026-01-01
Oligosaccharides, polymers of covalently linked monosaccharides, are essential biomolecules mediating diverse biological and physiological processes. Despite their critical roles, the structural complexity of many oligosaccharides poses a notable challenge for comprehensive characterization, limiting our understanding of their biological functions. Nanopores as emerging single-molecule sensors offer promise for oligosaccharide structural analysis. Here, we demonstrate direct discrimination of diverse mono- and oligosaccharides using a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore integrated with a single phenylboronic acid (PBA) adapter (MspA-90PBA). The high-resolution event signatures generated by MspA-90PBA enable structural analysis of complex oligosaccharides via hydrolysis into smaller fragments, which can be analyzed for either structure profiling or determination. Using a minimized reference database, we achieve structural determination of raffinose, stachyose, and verbascose through nanopore characterization of its hydrolyzed fragments followed by fragment assembly. This work establishes a framework for nanopore-based oligosaccharide analysis, combining single-molecule sensing with a fragment assembly strategy to address the long-standing challenge of glycan structural elucidation.