面向糖蛋白质组学的有机共价骨架富集新介质与方法

As one of the most common post-translational modification (PTM) types, reversible protein glycosylation plays crucial regulatory roles in almost all aspects of cellular processes, which is vital to tumor cancer, immune response and cell communication. At present, a group of novel glycopeptide enrichment materials were emerged to effectively solve the selective enrichment of sialylated glycopeptides. However, the remaining problems such as the specific enrichment of O-linked glycopeptides or neutral glycopeptides are still the core of the field. Thus, developing new generation of separation materials is quite urgent for promoting the enrichment accuracy and capture efficiency. In this project, covalent organic frameworks (COFs) provide continuous and confined open channels at the nanoscale, affording an accessible high surface area interface for modification. Functional units including amino acids, oligopeptides, and oligosaccharides are introduced to graft onto the surface of COFs hollow sphere, in order to the design of new organic functional enrichment materials. COFs has the advantage of chemical stability, customizable surface characteristics, and pore environment control. The ability to easily tune the construction unit of a COF using monomers bearing specific functional groups can improve its selective recongnition and highly compatibility with glycopeptides, in combination with the multiple hydrogen bonding sites, and the multi-cell collaborative interaction in the pore channels. Meanwhile, the selective enrichment method for glycopeptides will be established and validated with cell lysis buffer and serum samples. Based on these, we will do some exploratory research in areas like abnormal expression in intestinal cancer, and try to establish the relationship between glycosylation and disease. We believe this project will have important scientific significance and potential application value in the field of glycoproteomics, and provide an original strategy for the glycopeptide enrichment.

蛋白糖基化是生命科学研究的前沿方向，与肿瘤癌症发生，免疫应答，细胞通讯等密切相关。新型糖肽富集材料的开发很好地解决了唾液酸型糖肽富集的难题，然而对于O-连接糖肽、中性糖肽的特异性富集，仍是一个巨大的挑战。迫切需要引入新的材料学体系，提升富集的精确性和捕获效率。本项目以共价有机骨架材料（COFs）为载体，在COFs上引入氨基酸、寡肽、寡糖等功能识别单元，构造全新的有机功能富集材料。COFs具有化学稳定性好、结构裁剪性强、孔结构精细可调等优点，同时借助功能识别单元的多重氢键位点，以及孔内多单元协同的相互作用，实现对糖肽的选择性识别与高亲和力结合。进而构建材料对糖肽富集方法学，借助细胞裂解液、血清等实际样品进行验证。探索材料对肠癌组织样本中糖肽的捕获能力，构建糖基化表达异常与疾病之间的关系。COFs基新介质的引入，有望为糖肽的特异性富集和分离提供一种新方法、新思路。

蛋白质糖基化是生物体中最重要的翻译后修饰手段之一，糖蛋白/糖肽的有效分离和富集成为目前糖蛋白组学研究的首要问题。对于复杂的生物样本，糖蛋白的数量较少，酶解后大量高丰度非糖基化修饰肽的存在，使得低丰度糖肽的检测更加困难。因此，需要一些手段来有效地富集糖肽以提高其检测丰度，发展高选择性的糖肽富集材料及方法就成为在分子水平上有效地监测糖蛋白或糖肽的重要途经。相对于传统的糖肽富集材料，共价有机骨架材料具有比表面积大和可修饰位点丰富的优点，在糖肽富集领域具有很大的应用潜力。本项目制备了新型的共价有机骨架材料，分别利用不同单体通过共聚缩合反应生成的席夫碱构成了材料的框架，并采用COFs后修饰策略，从而提高材料的富集性能。利用扫描电镜、透射电镜、红外光谱和固体核磁等表征技术对材料的结构进行了表征，并将其应用于糖肽的选择性富集。分别对富集过程的上样条件、淋洗条件、洗脱条件进行富集条件的优化，从人血清免疫球蛋白G酶解液中观察到32个明显的糖肽信号峰。通过模拟复杂样本体系验证材料富集选择性，在人血清免疫球蛋白G和牛血清白蛋白的酶解液混合物中仍然保持了良好的选择性。此外，还考察了材料的检测限、富集容量、回收率等富集性能及在人血清等实际样品中的应用潜力，表现出较低的检测限，较高的富集容量，及较好的富集回收率。表明COFs材料在糖肽富集领域较好的应用前景，同时为糖肽的特异性富集和分离提供了一种新方法、新思路。