Profiling O-Acetylation in Sialoglycans Using MALDI-TOF and LC-MS/MS with Methylamidation and Permethylation...

O-acetylation of sialoglycans plays a critical role in modulating the structural and functional properties of glycoproteins, with implications in immune response, cell signaling, and disease pathology. Among sialic acids, N-acetylneuraminic acid (Neu5Ac) and its derivatives have garnered attention as biomarkers for various cancers and inflammatory conditions due to their terminal positioning on glycan chains and their susceptibility to biochemical modification. Despite their biological relevance, comprehensive analysis of O-acetylation patterns remains challenging due to their chemical lability and loss during conventional glycomic workflows, such as permethylation. This protocol outlines a robust workflow combining methylamidation and permethylation with porous graphitic carbon (PGC)-based purification and high-resolution MALDI-MS and LC-MS/MS to analyze O-acetylation in serum/plasma-derived N-sialoglycans. We demonstrate sensitive and reproducible profiling of sialylated glycan structures. This method enables deeper insight into glycan modification dynamics and can be broadly applied to biomarker discovery and comparative glycomics.