Analysis of <i>O-G</i>lycans 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 <i>N</i>-glycosylation, <i>O</i>-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 <i>O</i>-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 <i>O</i>-acetylated sialic acid residues, thereby facilitating the identification of <i>O</i>-glycans and their attached amino acid residues, as well as the determination of the number of <i>O-</i>acetyl groups. A total of 65 <i>O-</i>glycans can be identified from bovine mucin. Furthermore, the analytical strategy revealed that <i>O-</i>acetylated <i>N</i>-acetylneuraminic acid (Neu5Ac)-containing <i>O-</i>glycans from horse serum exhibited distinctive fragmentation patterns in comparison to those from bovine mucin. Additionally, the presence of deaminoneuraminic acid (KDN)-containing <i>O-</i>glycans was successfully confirmed in fish intestinal tissue. These findings suggest that this method provides an economical and potentially valuable tool for large-scale <i>O-</i>glycosylation studies in complex biological samples.