Analysis of O-Glycans by Oxidative Release Combined with 3-Nitrophenylhydrazine Derivatization

Glycosylation profiling is an effective methodology for achieving a comprehensive understanding of glycoproteins and their alterations in a multitude of pathological conditions. However, in comparison to N-glycosylation, O-glycosylation presents significant challenges in terms of both qualitative and quantitative mass spectrometric analyses. A recently developed oxidative release protocol enables the selective formation of O-glycans containing a carboxyl group derived from the amino acid residue. In this study, 3-nitrophenylhydrazine was used to derivatize the common carboxyl group in a mild hydrophilic solution. Derivatization resulted in the generation of a series of report ions for serine, threonine, sialic acid, and O-acetylated sialic acid residues, thereby facilitating the identification of O-glycans and their attached amino acid residues, as well as the determination of the number of O-acetyl groups. A total of 65 O-glycans can be identified from bovine mucin. Furthermore, the analytical strategy revealed that O-acetylated N-acetylneuraminic acid (Neu5Ac)-containing O-glycans from horse serum exhibited distinctive fragmentation patterns in comparison to those from bovine mucin. Additionally, the presence of deaminoneuraminic acid (KDN)-containing O-glycans was successfully confirmed in fish intestinal tissue. These findings suggest that this method provides an economical and potentially valuable tool for large-scale O-glycosylation studies in complex biological samples.