选择性分离和富集代谢标记的糖蛋白

Glycosylation is one of the most common and important post-translational modification of the protein, and plays crucial role in many biological events, involved in cell- cell adhesion, recognition and signaling. The diversity and complexity of the micro-structures are related to many diseases and tumor metastasis. Heterogeneity hampered the progress toward understanding the functions of the oligosacccharidses. It is a big chanllenge to enrich and separate the glycans and glycoproteins. In this proposal, we develop an new strategy for enrichment and separation of the fucosylted and sialylated glycoprotein.Understanding the microstructure of sialic acid and fucose has enormous potential for understanding the biological functions and treating diseases. This strategy is based on the combination of metabolic oligosaccharides engineering and nano-technology. The aims are 1)designing, synthesizing new unnatural sialic acid/fucose with chemical reportor-aldehyde, 2)incorporating the unnatural sugar into the cell surface by metabolic engineering, 3) synthesizing and characterizing new functionalized magnetic nano-particle, 4)taging the magnetic nanoparticel onto the cell surface by oxime ligation, 5)enriching and separating the taggd glycopretein, releasing and analyzing the glycoproteins. In this application, we propose to establish a standard method to enrich and separate sialylated and fucosylated glycoproteins: systemactially evaluate the bio-compatability of the synthsized sugar; evaluate and optimize the property of the nanoparticles,optimize the condition of the enrichment and separtion methods. It is anticipated that the proposed research will establish an standard method of enrichment and separation of the two types glycoprotein, accelarate the study the micro-structure of the glycomes, benefit the understanding of their biofunction and related diseases.

糖蛋白上糖链的分支结构以及表达的密度对细胞-细胞间的信号传递，细胞的复制等细胞功能有着举足轻重的作用。糖蛋白微观结构的改变与多种疾病和癌症的发展和转移有着明显的关系，对岩藻糖基化蛋白和唾液酸化糖蛋白的研究对癌症的早期检测和诊断，癌症的介入治疗都有明显的参考意义。在不对细胞的遗传物质进行操作的情况下，利用表达在细胞表面有化学报告基团的非天然糖，通过特殊的化学反应对其进行修饰。本项目将在前期研究的基础上，以岩藻糖和唾液酸为结构基础，以代谢寡糖工程技术为手段，对活体细胞表面的岩藻糖基化蛋白和唾液酸化糖蛋白进行研究。综合运用磁性纳米粒子，对其进行分离和富集，再使用LC-MS，MS， NMR等技术对其进行分析，分析活体细胞该糖蛋白的微观结构及其变化。本项目将为岩藻糖蛋白的生理作用和临床应用提供科学依据。

生命体内蛋白质上的糖链结构的微观不均一性，极低的含量，使检测和跟踪糖蛋白成为糖科学研究中待解决的重要基础性问题之一。磁性纳米粒子作为一种新型的纳米材料，具备纳米粒子的化学和物理性能，并且有着独特的磁学性质，能在外加磁场下迅速分离，且磁性纳米粒子表面能进行各种修饰，不仅丰富了磁性纳米材料的种类，也能为生物分析领域提供新的材料。本课题的工作研制了含不同可裂解的功能基团的磁性纳米粒子，包括含可断裂的氨氧基，可断裂的叠氮基和含吡啶二硫功能基的磁性纳米粒子，并进行了形貌分析。本课题利用含氨氧基磁性纳米粒子对Jurkat细胞表面上的含唾液酸的糖蛋白进行分离和富集，并利用LC-MS/MS进行了组学分析，在组学分析里中，根据得到的48687种肽段，并根据肽段推测得到2371种已知蛋白，且其中无岩藻糖基化的N-链接糖肽47种。目前已接受文章一篇，已申请专利1项,一篇文章已投递。另外两篇文章和两项专利正在准备中。项目目前取得的经验为以后制备磁性纳米粒子对进行广谱、定量和都对比糖蛋白组学研究打下了基础。