Differentiation of α2,3- and α2,6-linked sialylated N/O-glycan isomers in human seminal plasma by an glycoqueuing strategy

N/O -glycans in various human seminal plasma (hSP) glycoproteins, especially α2,3- and α2,6-sialylation levels, are closely associated with semen quality. However, effective differentiation of sialyl linkage isomer remains unachieved. Here, we employed our previously developed glycoqueuing strategy for isomer-specific quantitative analysis of sialylated N/O- glycans released from hSP. A total of 21 exclusively α2,6-sialylated and 14 α2,3-sialylated (bearing α2,3-linked sialic acids) N- glycan isomers were detected, and the relative abundance of α2,6-sialylation among these sialylated N -glycans reached 61.40 %. For O -glycans, seven monosialic and five disialylated species were observed, and all were confirmed to be α2,3-sialylated. Nonsialylated N/O- glycan isomers were simultaneously quantified via hydrophilic interaction liquid chromatography-tandem mass spectrometry. Specifically, 21 nonsialylated N- glycan isomers were identified, among which the relative abundances of oligomannose-type and complex-type glycan were each approximately 50 %. All O- glycans exhibited core 1 or core 2 structures, including 12 α2,3-sialylated and 26 nonsialylated species. Notably, four isomers separated from two newly discovered nonsialylated O -glycan compositions H2N1F1 and H2N1F2 (H: hexose, N: N- acetylgalactosamine, F: fucose) were identified in hSP. Additionally, sialylated and nonsialylated N/O- glycans were all highly fucosylated (16.98 %–67.92 %), and bore numerous Lewis X and Lewis Y structures. The detailed glycan structural and distribution data, particularly α2,3/α2,6-sialylation profiles, not only provides a reference for constructing the hSP glycomic fingerprint, but also supports in-depth investigation of hSP glycan functions in reproduction and exploration of novel glycan markers for clinical semen quality detection.