摘 要

糖基化是真核生物蛋白质中最普遍的翻译后修饰之一，在众多生物进程中起到至关重要的作用。对糖链进行准确、高效分析是了解糖基化过程、掌握疾病机理的重要基础。

本研究中，我们发展了一种高效、高选择性质谱法分析N-糖链策略,实现了生物样品中糖链的高选择性、高灵敏度检测。具体内容如下：

1. 选用新型氟化碳标签C₁₁H₁₁F₁₃N₂O₂为标记物，对N-糖链进行了成功标记；
2. 通过氟固相萃取，对标记后N-糖链进行了有效富集纯化；
3. 将衍生化结合氟固相萃取技术应用于生物样品的检测，在5 μg人血清样品中成功解析出12种N-糖链结构。

本研究结果表明，氟标记结合氟固相萃取柱技术可以直接对酶解后复杂生物样品进行高选择性、高灵敏度糖链分析，减少了样品消耗，省去了其它繁琐的样品处理步骤，为以后生物样品糖链分析、寻找疾病标志物奠定了坚实的基础。

关键词：糖链；氟化标记；氟固相萃取；基质辅助激光解吸电离飞行时间质谱法

Abstract

Glycosylation is one of the most common post-translational modifications in eukaryotic proteins and it plays a crucial role in many biological processes. Hence, the analysis of glycans is very necessary for the understanding of glycosylation processes and the clarification of the mechanisms of many diseases.

In this study, an efficient strategy has been developed for N-glycan analysis via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The contents of this thesis are summarized as follows:

(1) A fluoride carbon tag (C₁₁H₁₁F₁₃N₂O₂) was successfully labeled with N-glycans by oxime formation;

(2) The derivatized glycans were efficiently purified by fluorous solid-phase extraction (F-SPE);

(3) Derivatization combined with F-SPE was applied for the analysis of human serum samples, and more than 12 N-glycans were indentified.

In conclusion, a two-step strategy, including fluoride carbon tagging and F-SPE, was successfully applied for N-glycans analysis from biomixtures. A novel fluoride carbon tag was, for the first time, used to label N-glycans by oxime formation reaction and showed high derivatization efficiency. The F-SPE after tagging showed great selectivity towards derivatized glycans, avoiding the conventional tedious sample preparation procedures and facilitating the N-glycan analysis with high sensitivity and low sample consumption.

Key Words：Glycans；Fluorinated Tag；Fluorous Solid-Phase Extraction；Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

目 录

第1章 绪论 1

1.1 糖组学概述 1

1.1.1 蛋白质糖基化修饰的作用 1

1.1.2 糖蛋白/糖链类型 2

1.2 聚糖分析方法 3

1.2.1 毛细管电泳法 3

1.2.2 核磁共振法 4

1.2.3 高效液相色谱法 4

1.2.4 质谱法 4

1.3 糖链质谱检测方法 7

1.3.1 糖链的释放方法 7

1.3.2 蛋白质变性 7

1.3.3 糖链的衍生方法 8

1.3.4 衍生产物的纯化分离 9

1.3.5 MALDI-TOF质谱检测 10

1.4 结论 10

第2章 氟化碳标记与模型聚糖衍生反应 12

2.1 引言 12

2.2 实验部分 12

2.2.1 材料与试剂 12

2.2.2 仪器设备 13

2.2.3 实验步骤 14

2.3 结果与讨论 14

2.3.1 DP7与PFHA衍生化反应 14

2.3.2 DP7衍生条件优化 15

2.3.3 衍生化DP7高灵敏度分析 17

2.4 结论 18

第3章 N-糖链的氟固相萃取 19

3.1 引言 19

3.2 实验部分 19

3.2.1 材料与试剂 19

3.2.2 仪器设备 19

3.2.3 实验步骤 20

3.3 结果与讨论 22

3.3.1 缓冲溶液对酶切效率的影响 22

3.3.2 RNase B糖链衍生条件再优化 23

3.3.3 F-SPE纯化富集衍生糖链条件优化 24

3.3.4 F-SPE纯化富集效率评价 26

3.3.5 衍生化结合氟固相萃取在生物样品中的应用 28

3.4 结论 28

第4章 总结与展望 30

参考文献 31

致谢 35