Differentiation of α2,3- and α2,6-Linked Sialylated N/O-Glycan Isomers in Human Seminal Plasma by an Improved Glycoqueuing Strategy

Abstract

Abstract N/O-glycans in human seminal plasma (hSP) proteins regulate semen adhesion and liquefaction, sperm capacitation, acrosome reaction, and sperm-egg fusion. Given that different glycan isomers in hSP, such as α2,3- or α2,6-sialylated N/O-glycans, determine different activities, yet their differentiation have not been realized so far. We employed our previously improved glycoqueuing strategy for isomer-specific quantitative analysis of sialylated N/O-glycans released from hSP. A total of 23 α2,6- and 12 α2,3-sialylated N-glycan isomers were detected, and the relative abundance of α2,6-sialylation was 61.40%. Seven monosialic and five disialylated O-glycans were observed, and all of them were α2,3-linked. Nonsialylated N/O-glycan isomers were also quantified by hydrophilic interaction liquid chromatography-tandem mass spectrometry. Twenty-one nonsialylated N-glycan isomers were detected, of which half were of the oligomannose or complex type, with only trace amounts of hybrid glycans. All 26 nonsialylated O-glycans were core I or II structures, and the four isomers of H2N1F1 and H2N1F2, composed of hexose (H), N-acetylgalactosamine (N), and fucose (F), were found for the first time. Sialylated and nonsialylated N/O-glycans were highly fucosylated (16.98–67.92%). These detailed structure and distribution information will contribute to further research on the molecular mechanism of hSP glycan components regulating reproductive processes.