Dong Xue, Zhou Shiyue, Mechref Yehia
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
Electrophoresis. 2016 Jun;37(11):1532-48. doi: 10.1002/elps.201500561. Epub 2016 Apr 29.
Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers.
牛奶中的寡糖不仅为婴儿提供营养，还具有重要的免疫生物功能，如抑制病原体与宿主细胞结合。牛奶寡糖的主要成分是游离寡糖。由于牛奶中的蛋白质高度糖基化，牛奶中的N-聚糖也起着重要作用。在本研究中，我们使用液相色谱-串联质谱法（LC-MS/MS）研究了从牛、山羊和人乳中提取的全甲基化游离寡糖和N-聚糖。报告了游离寡糖和N-聚糖的定量概况。在牛、山羊和人乳样品中观察到的游离寡糖数量（不考虑异构体）分别为11、8和11。人乳中的游离寡糖结构比其他两种乳样品更为复杂。分别有58、21和43种N-聚糖结构（不考虑异构体）与从牛、山羊和人乳样品中提取的乳清蛋白相关。牛乳铁蛋白中的游离寡糖和N-聚糖高度唾液酸化，岩藻糖基化程度较低。山羊乳和人乳铁蛋白中的游离寡糖和N-聚糖均高度岩藻糖基化。此外，通过高温下的多孔石墨化碳液相色谱法测定了乳样品中的异构体聚糖。例如，使用多孔石墨化碳柱实现了人乳游离寡糖Gal-GlcNAc-(Fuc)-Gal-Glc和Gal-GlcNAc-Gal-Glc-Fuc异构体的分离。聚糖结构的全甲基化有助于串联质谱的解析。例如，在全甲基化聚糖的串联质谱中，内部裂解和糖苷键裂解很容易区分。这一特性导致了几种异构体的鉴定。
