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

高等学校化学学报 ›› 2010, Vol. 31 ›› Issue (11): 2173.

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

薛向东1, 张萍1,2, 王仲孚1, 黄琳娟1

1. 西北大学生命科学学院, 西部资源生物与现代生物技术教育部重点实验室, 西安 710069;
2. 咸阳师范学院化学与化工学院, 咸阳 712000

收稿日期:2010-04-15 出版日期:2010-11-10 发布日期:2010-11-10
通讯作者: 黄琳娟, 女, 教授, 博士生导师, 主要从事糖生物学与糖工程学研究. E-mail: huanglj@nwu.edu.cn
基金资助:
国家“八六三”计划项目(批准号: 2007AA10Z338, 2007AA091601)、国家自然科学基金(批准号: 30870548)和教育部新世纪优秀人才支持计划(批准号: NCET-08-0893)资助.

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

XUE Xiang-Dong1, ZHANG Ping1,2, WANG Zhong-Fu1, HUANG Lin-Juan1*

1. Key Laboratory for Western China Resource Biology and Biotechnology, Ministry of Education, College of Life Sciences, Northwest University, Xi′an 710069, China;
2. School of Chemistry and Chemical Engineering, Xianyang Normal University, Xianyang 712000, China

Received:2010-04-15 Online:2010-11-10 Published:2010-11-10
Contact: HUANG Lin-Juan. E-mail: huanglj@nwu.edu.cn
Supported by:
国家“八六三”计划项目(批准号: 2007AA10Z338, 2007AA091601)、国家自然科学基金(批准号: 30870548)和教育部新世纪优秀人才支持计划(批准号: NCET-08-0893)资助.

摘要/Abstract

摘要： 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.

关键词: 苯胺稳定同位素标记, 电喷雾电离质谱, 还原氨化, 寡糖的定量

Abstract: 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.

Key words: Aniline stable isotopic labeling, ESI-MS, Reductive amination, Quantitation of oligosaccharide

TrendMD:

薛向东, 张萍, 王仲孚, 黄琳娟. 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. 高等学校化学学报, 2010, 31(11): 2173.

XUE Xiang-Dong, ZHANG Ping, WANG Zhong-Fu, HUANG Lin-Juan* . Analysis Method for Relative Quantitation and Qualitation of Reductive Glycans via Aniline Stable Lsotopic Labeling and ESI-MS. Chem. J. Chinese Universities, 2010, 31(11): 2173.

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