Highly Efficient Identification of O-GalNAc Glycosylation by an Acid-Assisted Glycoform Simplification Approach

Compared with N-linked glycosylation, the analysis of O-GalNAc glycosylation is extremely challenging due to the high structure diversity of glycans and lack of glycosidases to release O-GalNAc glycans. In this work, a glycoform simplification strategy by combining HILIC enrichment with chemical de-sialylation to characterize O-GalNAc glycosylation of human serum is presented. This method is first validated by using the bovine fetuin as the test sample. It is found that more than 90% of the sialic acid residues can be removed from bovine fetuin by the acid-assisted de-sialylation method, which significantly simplifies the glycan structure and improves identification sensitivity. Indeed, the number of identified peptide backbones increases nearly one fold when this strategy is used. This method is further applied to analyze the human serum sample, where 185 O-GalNAc modified peptide sequences corresponding to 94 proteins with high confidence (FDR (false detection rate) <1%) are identified. This straight forward strategy can significantly reduce the variations of glycan structures, and is applicable to analysis of other biological samples with high complexity.