Oxidative Release of Natural Glycans: Unraveling the Mechanism for Rapid N-Glycan Glycomics Analysis.

N-glycosylation is a critical post-translational modification involved in various biosynthetic pathways and disease mechanisms. In this study, we present an optimized oxidative release of natural glycans (ORNG) method using household bleach that enables the rapid and efficient release of N-glycans from biological samples. We thoroughly investigated the ORNG mechanism, identifying key intermediates and side products and providing valuable insights into the oxidative release process. The method is highly efficient, releasing a wide range of N-glycans, including high-mannose, hybrid, and complex structures, with minimal sample processing. Our ORNG-based specific N-glycan profiling approach has demonstrated high sensitivity and efficiency, particularly in releasing N-glycans resistant to enzymatic digestion, such as core α3-fucosylated N-glycans from soy protein. Validation through mass spectrometry confirmed the method's ability to accurately profile N-glycans from complex biological samples, including human serum, with results comparable to traditional PNGase F digestion. The ORNG-based method's scalability, versatility, and use of low-cost reagents make it especially suited for large-scale glycomics studies. Furthermore, the mass spectrometry data revealed that the ORNG-based method achieves high sensitivity and specificity, positioning it as a robust alternative for comprehensive glycan profiling and functional studies. Our findings highlight ORNG's potential to advance N-glycomics, offering promising improvements in speed, efficiency, and breadth of glycan analysis.