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

Characterization of O -glycans linked to serine or threonine residues in glycoproteins has mostly been achieved using chemical reaction approaches because there are no known O -glycan-specific endoglycosidases. Most O -glycans are modified with sialic acid residues at the non-reducing termini through various linkages. In this study, we developed a novel approach for sialic acid linkage-specific O -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 derivatization of ethyl-esterified O -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-specific N - and O -linked glycan analyses of a model glycoprotein and human cartilage tissue. This novel glycomic approach will facilitate detailed characterization of biologically relevant sialylated N - and O -glycans on glycoproteins.