Simultaneous and sialic acid linkage-specific N- and O-linked glycan analysis by ester-to-amide derivatization

In this study, we developed a novel approach for sialic acid linkage-specificO-linked glycan analysis through lactone-driven ester-to-amide derivatization combined with non-reductive β-elimination in the presence of hydroxylamine.O-glycans released by non-reductive β-elimination were efficiently purified using glycoblotting via chemoselective ligation between carbohydrates and a hydrazide-functionalized polymer, followed by modification of methyl or ethyl ester groups of sialic acid residues on solid-phase. In-solution lactone-driven ester-to-amide derivatiza tion of ethyl-esterifiedO-glycans was performed, and the resulting sialylated glycan isomers were discriminated by mass spectrometry. In combination with PNGase F digestion, we carried out simultaneous, quantitative, and sialic acid linkage-specificN- andO-linked glycan analyses of a model glycoprotein and human cartilage tissue. This novel glycomic approach will facilitate detailed characterization of biologically relevant sialylatedN- andO-glycans on glycoproteins.
Source: Glycoconjugate Journal - Category: Biochemistry Source Type: research