通用的化学法释放糖蛋白N/O-糖链及其高灵敏度质谱分析新方法研究

Generally, release of glycan from skeleton protein by a suitable and efficient method is especially essential to elucidate their structure. However, the existed methods for release N, O-glycans from glycoprotein suffer from some limitations and drawbacks. Hence, the detection of N,O-glycosylations has been hindered to some extent. In our previous work, we have preliminarily established and reported an innovative route for non-reductive release and analysis of O-glycans from glycoprotein. Our current studies will systematically examine and evaluate the alkali catalytic N, O-glycan release process according to the structure and stability difference of N/O- glycopeptide bond under alkali conditions, and establish a universal and non-reductive release methods for specific O- or N-glycans as well as both O-and N-glycans. The studies will emphatically focus on establishment of the novel and unified approach for O/N-glycan liberation and prevent peeling degradation in the process of release procedure. Furthermore, with the strengths of mass spectrometry and liquid chromatography -mass spectrometry technique, we will employ the develped release method that could release N-, O-, and N/O-glycans from glycoprotein and show compatibility with subsequent permethylation, to establish an high sensitive analysis methods for the detection of N/O-glycans and provide an avenue to build a tandem MS fragment database of glycans. Our studies will improve the existing methods for microscale N/O-glycan releasing, labeling, capturing, qualitative and quantitative detection, which will definitely facilitate the development of high throughput sequencing glycans, illustrating the methological significance in structure-function relationship studies.

研究糖蛋白糖链结构，通常需要将糖链从糖蛋白上释放下来。由于现有释放N，O-糖链的方法具有局限性，影响了后续分析鉴定。本课题拟在我们已初步建立的O-糖链非还原性释放方法基础上，进一步深入系统研究碱催化的N，O-糖链释放过程，根据N和O-糖肽键及结构类型不同的糖链在碱性环境中稳定性不同，按分析需要选择不同条件，在同时存在N和O-糖基化情况下，实现特异性高效定量释放N或O-糖链，或N,O-糖链同时释放，重点解决糖链在释放过程中的降解问题，进而发展通用的N,O-糖链释放方法体系；以质谱和液-质联用技术为检测手段，建立基于释放基础之上的糖链高灵敏度分析鉴定方法，对PMP标记的糖链全甲基化后的质谱碎片裂解行为进行研究，构建糖链碎片图谱库。本课题研究将进一步完善和改进现有微量糖链释放、标记、富集及定性定量分析方法体系，对发展新的高通量糖链序列结构分析策略有重要意义，也将为糖链结构与功能关系研究提供手段。

研究糖蛋白糖链结构，通常需要将糖链从糖蛋白上释放下来。由于现有释放N，O-糖链的方法具有局限性，影响了后续分析鉴定。项目自立项后，本课题组根据课题的研究任务，认真组织研究任务的落实，经过4年的努力，已经全面完成研究计划，取得了一些重要的创新性成果：1. 改进和完善了糖蛋白O-糖链的非还原性释放条件，完全防止糖链的降解（peeling）反应。2. 改进和完善了含有核心α-1, 3岩藻糖连接的N-糖链的释放条件，防止样品糖链的降解（peeling）反应。3．建立了选择性的释放N-或O-糖链反应条件的研究。4. 建立了碱性介质中N, O-糖链同时释放反应条件。5. 建立了释放后糖链衍生物的HPLC及LC-MS分离分析方法的及同分异构体的分离。6. 建立了1-苯基-3-甲基-5-吡唑啉酮（PMP）标记的N, O-糖链全甲基化后的质谱碎片裂解行为。7.将建立的方法用于人乳结合型寡糖等复杂生物样品糖蛋白糖链的研究，取得了较好的研究结果。本课题研究结果对发展新的高通量糖链序列结构分析策略有重要意义，也将为糖链结构与功能关系研究提供手段。