Native PGC-LC–MS profiling reveals distinct O-acetylation patterns of Sialylated N-glycans across mammalian sera

O-acetylation of sialic acids represents an additional layer of structural diversity and biological complexity, occurring at various hydroxyl positions (commonly C-7, C-8, or C-9) of the sialic acid residue. This modification modulates the recognition of sialylated glycans by lectins, antibodies, and viral proteins, and contributes to viral tropism and host susceptibility, particularly in influenza and coronaviruses that bind O-acetylated sialylated receptors. However, current LC-MS glycomics workflows commonly employ reduction or permethylation, which, while improving chromatographic stability and ionisation, result in the loss of labile O-acetyl groups, obscuring their biological relevance. Native glycan analysis, in contrast, preserves the complete structural integrity of glycans, enabling accurate detection of labile modifications. Using a native released glycan workflow limited to pH ≤ 8, O-acetylated N-glycans were detected in mouse and rat sera that were previously undetectable under basic derivatisation conditions. Beam-type collision-induced dissociation generated the most informative fragmentation spectra, with diagnostic ions confirming O-acetylated NeuGc and NeuAc residues. Chromatographic profiling revealed later elution and broadened peak shapes for O-acetylated species, consistent with increased hydrophobicity and microheterogeneity. A checkpoint-based identification workflow incorporating isotopic, chromatographic, and MS2 criteria reduced false positives, retaining only 3-5% of putative O-acetylated glycans as confident identifications. Quantitative comparison across species revealed extensive O-acetylation in rat (53.4%) and moderate modification in mouse (8.8%), but none detectable in human serum. These findings establish a robust analytical framework for native detection and characterisation of O-acetylated N-glycans, revealing species-specific regulation of this labile modification.