Comparison results for each method.

Glycan structures hold promise as biomarkers for the early detection of diseases, owing to their sensitive reflection of cellular states. However, glycan analysis remains complex and time-consuming, and the use of hazardous chemicals in traditional hydrazinolysis methods presents a significant barrier to broader application and cross-disciplinary research. To enable efficient glycan biomarker discovery, we aimed to develop a simple and accurate N-glycan analysis method capable of high-throughput sample processing. A key feature of this study is the use of pyridylamination, a fluorescent labeling technique that enables high isomer separation efficiency in reversed-phase LC/MS. After comparing various methods for N-glycan release and purification, we identified Rapid PNGase F (New England Biolabs) and BlotGlyco (Sumitomo Bakelite) as optimal for this application. To improve accuracy by reducing artifact formation, the BlotGlyco protocol was modified from the manufacturer’s original instructions. Using this optimized workflow, we successfully analyzed human serum, human urine, and CHO-K1 membrane fractions, demonstrating that distinct glycan structures in each sample type could be effectively separated and quantified. This work supports the broader adoption of glycan analysis across diverse research fields.