Glycomics serves as a method to define the sugar-based molecules in a given sample, and is complementary with database-based proteomics studies, by defining the modifications to the proteome. Historically, glycan analysis has involved high pH treatment to make glycans more amenable to analysis, including reduction and permethylation. This high pH treatment can result in the loss of labile modifications such as O-acetylation, which are key in host-pathogen communication. Challengingly, the loss of these labile modifications leave no molecular evidence, hampering the ability to characterise the true glycome.
Here, we describe a native glycomics method that stabilises these 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 in typical glycomics workflows. Furthermore, an oxidative release at pH 7 releases O-acetylated O-glycan acids, enabling their detection without the typical high-pH beta-elimination sample preparation. 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.
We reproduced the findings of Liu et al. which analysed complementary intact glycopeptides. Using the glycan modification space defined in our glycomics study, we detected greater than 50% sialic acid O-acetylation in rat serum and approximately 5% in mouse serum, with no evidence of O-acetylation in human serum. These results demonstrate strong concordance between released glycan and glycopeptide analyses, underscoring the reproducibility and cross-platform validity of native O-acetylation measurements. These findings establish a robust analytical framework for native detection and characterisation of O-acetylated glycans, revealing species-specific regulation of this labile modification.