Nanopore profiling and structure determination of oligosaccharides by fragmentation

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.