基于液质联用技术的糖蛋白O-糖链结构快速完全解析新方法研究

As an important type of informative biomolecule, O-glycans of glycoproteins have a series of biofunctions. High-throuput structural analysis is the basis for systematic investigations on the correlations between their structure and functions. However, it is still a challenge to elucidate their structural details completely and rapidly, due to the limitations of those already developed analytical techniques. Therefore, this program plans to develop a novel strategy for rapid and complete structural elucidation of complex biological sample O-glycans by liquid chromatography coupled with mass spectrometry (LC-MS) analytical methods with a structure encoding library of structurally known O-glycans as a standard reference. First of all, a method for high-resolution structural differentiation of O-glycans will be developed, based on the combination of online hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry and online reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry. Subsequently, a series of structurally known glycoprotein O-glycans will be analysed in detail with both the LC-MS methods, and the obtained data that are related with glycan structure and their corresponding O-glycan structure schemes will be collected and organized to form a standard O-glycan structure encoding library (SOGSEL). Finally, the O-glycans derived from unknown samples will be analysed in both the LC-MS modes and the obtained analytical data will be retrieved in the SOGSEL to get their corresponding complete O-glycan structure. This new strategy can overcome the technical defects of traditional glycan structure analysis methods such as complicated operation, repeated analysis as well as low efficiency. Therefore, it has an important significance in the methodological studies on high-throughput sequencing of glycoprotein O-glycans, and can lay a technical foundation for standardized, systematic, and large-scale analysis of biological O-glycomes.

糖蛋白O-糖链有多种生物功能，是重要的生物信息分子，高通量化结构分析是系统研究其结构与功能之间关系的前提。但是，由于现有技术的局限性，对O-糖链结构进行快速完全解析仍然具有很大的挑战性。本项目拟以亲水液相色谱-串联质谱在线联用与反相液相色谱-串联质谱在线联用联合分析技术为手段，建立O-糖链结构的高分辨区分方法，并进一步对各种结构已知的糖蛋白O-糖链进行分析，利用所得结构相关分析参数与糖链结构之间的一一对应关系构建O-糖链标准结构编码库，发展以结构编码库为参照标准对复杂生物样品O-糖链结构进行快速完全解析的新策略。该方法的建立，可克服传统糖链结构分析方法操作复杂、重复分析、效率低下的缺点，对糖蛋白O-糖链高通量测序的方法学研究有重要意义，可为标准化、系统化、规模化的生物O-糖组分析奠定技术基础。

糖蛋白O-糖链是一类重要的生物信息分子，广泛参与细胞黏附、发育分化、信号转导等一系列生物过程，并与炎症、肿瘤、神经退行性疾病等多种疾病的发生发展密切相关，对其结构-功能关系的研究已成为揭示多种生物过程和疾病分子机制的关键。但是，由于技术上的限制，目前仍难以对各种O-糖链同分异构体的结构进行高分辨率、高通量化识别和解析，严重制约了O-糖链功能及相关疾病发生机理的研究。为解决这一问题，本项目基于液质联用技术发展了一种对O-糖链同分异构体结构进行快速解析的新策略。首先，对O-糖链不同模式HPLC分离条件进行了优化，建立了O-糖链PMP衍生物的高分辨率在线HILIC-MS/MS和RP-HPLC-MS/MS联合分析方法；然后，发展了糖链PMP衍生物的全甲基化修饰及MSn顺序裂解结构分析方法；接着，通过对不同分析条件下O-糖链相对于内标的保留时间的观测值进行统计学分析，证明了在LC-MS/MS分析中采用内标法对O-糖链结构进行统一数字化编码的可行性，并以几种典型的糖蛋白样品初步构建了一个O-糖链结构编码库；最后，成功将该编码库用于30多种复杂生物样品或糖蛋白样品中O-糖链结构同分异构体的快速鉴定。在研究过程中发现，除了极少数特殊情况之外，当色谱条件发生一定程度的变化时，O-糖链相对于内标的保留时间的相对标准偏差一般均小于2%，说明该值不会发生显著改变，这一结果充分证明了构建O-糖链结构编码库的可行性，从而为本研究奠定了关键基础。因此，该研究为O-糖链结构分析提供了一种新策略，对O-糖组学研究有重要意义。