基于电喷雾电离质谱(ESI-MS)的苯胺稳定同位素标记对还原性寡糖(链)的定性定量分析方法

建立了一种基于电喷雾电离质谱的苯胺稳定同位素标记对还原性寡糖链进行定性及相对定量分析的研究方法. 用苯胺标记乳糖标准品, 优化了影响标记效率的各种因素, 在弱酸性环境下, 选择糖链/苯胺/硼氢氰化钠的摩尔比为1∶1.2∶10, 于70 ℃反应15 min即可标记完全; 同时考察了4对d0/d5苯胺标记的麦芽糊精寡糖在电喷雾电离质谱中的线性、动态范围以及重现性. 结果表明, 在15倍动态范围内, 相对定量方法呈良好的线性关系(R=0.9986)和重现性(CV=10.20%). 为进一步验证定量方法的可靠性, 将其应用于人奶中游离寡糖(HMOs)和牛奶中游离寡糖(BMOs)的分析. 研究结果表明, 人奶中的乳糖含量高于牛奶, 人奶游离寡糖比牛奶游离寡糖种类复杂, 且岩藻糖基化程度高. 该方法成本低廉, 标记效率高且后处理方法简单方便, 适于微量样品通量化分析, 对差异糖组的研究有重要意义.

Analysis Method for Relative Quantitation and Qualitation of Reductive Glycans via Aniline Stable Lsotopic Labeling and ESI-MS

An established method for qualitation and quantitation of reductive glycans based on aniline stable isotopic labeling was reported. Various conditions which affect the derivatization efficiency were optimized. Reducing oligosaccharide could be quantatively derivatized by aniline at 70 ℃ within 15 min, under the condition that the saccharide, aniline and sodium cyanoborohydride were in a molar ratio of 1∶1.2∶10. In order to test the linearity and dynamic range of this method, aliquots of standard oligosaccharide(maltodextrin) were labeled with d0/d5-aniline separately. The results indicate that the isotopic labeling method employed here could provide relative quantitative data with a dynamic range of 15-fold, adequate linearity(R=0.9986) and reproducibility with a coefficient of variation that is 10.30% on average. The established method was sensitive and reproducible for the candidate glycans and was successfully used for comparing the differences between free milk oligosaccharides for human(HMOs) and bovine(BMOs). The results show that the lactose amount in human milk is higher than that from bovine milk, and the varieties of oligosaccharides from human milk are more complex than that of bovine milk. The relative quantitative and qualitative method established here will provide an alternative option for studying the changes of the glycans and further understanding the mechanism of the glycosylation, which associate with a wide assortment of diseases intimately.