Development of derivatization-enhanced EIEIO-driven glycosidic linkage sequencing (DEED-GL-Seq) strategy and its application to glycogen-sourced oligosaccharides profiling

The structural complexity arising from diverse glycosidic linkages in oligosaccharides hinders the elucidation of their biological functions. Existing analytical methods often lack the necessary feasibility and accessibility for comprehensive linkage analysis. To address this, we developed Derivatization-Enhanced EIEIO-Driven Glycosidic Linkage Sequencing (DEED-GL-Seq), a novel strategy leveraging differential electron density modulation around glycosidic bonds upon N²,N²,N⁴,N⁴-tetraethyl-6-hydrazineyl-1,3,5-triazine-2,4-diamine (T3) derivatization. This method integrates EIEIO MS² to identify glycosidic bond-specific diagnostic fragments for linkage determination. Validation confirmed DEED-GL-Seq's specificity, sensitivity, reliability, and standard-independence. Coupling with fine-tuned HILIC separation further enhanced its analytical power for complex matrices. We applied DEED-GL-Seq to profile oligosaccharides derived from partially hydrolyzed glycogen, validating our results against reference standards. By analyzing the abundance ratios of specific trisaccharides, we predicted the branching degrees of glycogen and amylopectin, corroborating existing literature. Notably, DEED-GL-Seq identified established (Glc₄) and novel (HEX-1, HEX-2, HEPTA-1, HEPTA-2) potential glycosidic biomarkers for GSD-II in urine, with the novel oligosaccharides showing superior diagnostic performance. The unique structure of HEX-2 suggests distinct biosynthetic pathways in GSD-II pathophysiology. DEED-GL-Seq represents a significant advancement in glycomics, offering a powerful tool for comprehensive oligosaccharide profiling and laying a foundation for in-depth functional investigations. This work presents a novel application paradigm for EIEIO technology.