基于高分枝聚合物材料富集糖肽新方法的研究

发布时间：2018-05-03 18:28

本文选题：高分枝聚合物 + 酰肼富集　；参考：《安徽医科大学》2014年硕士论文

【摘要】：蛋白质糖基化作为一种最常见的翻译后修饰。它不仅在蛋白质的稳定性、溶解性、折叠和功能执行方面起着关键的作用,而且参与细胞内蛋白质的运输、定位和分子识别。此外,异常糖基化也已经被证明和癌症等疾病相关,许多糖蛋白可以作为临床生物标志物和治疗靶标。因此,糖蛋白和糖肽的分离、发现和鉴定变得越来越重要,它可以帮助我们更好地理解生物过程,寻找潜在的诊断标志物和治疗靶点。依靠生物质谱对糖肽规模化定性与定量分析,已成为当前蛋白质翻译后修饰研究的一个重要研究方向。由于糖肽仅占所有酶解后肽段的小部分(2%～5%)[7],其质谱响应很容易被高丰度非糖肽抑制。另外,糖链的微观不均一性进一步降低了糖肽的相对量而使得它们难以被质谱检测。因此,对于糖肽的特异性富集至关重要。凝集素[8]和抗体[9]是最重要的两种亲和富集方法,但具有稳定性差,价格昂贵的缺点。分子排阻、亲水色谱、硼酸富集也都广泛应用于糖肽的富集。但是,各种富集手段都存在一定的缺点,这些都使得糖蛋白组学的研究进展相对于磷酸化、泛素化、乙酰化等其它翻译后修饰,更为落后。近年来,酰肼固相提取法作为传统的富集N-糖蛋白/糖肽的富集方法也备受关注。但是它也存在一些不可避免的缺点：样品损耗大,步骤繁琐。此外,最常用的商业化酰肼树脂不仅价格高,而且在溶液中结合糖蛋白/糖肽能力有限。因此,目前依靠酰肼富集N-糖蛋白/糖肽的方法还有待完善。高分枝聚合物由于其独特的结构特性,如准确的大小,内部空腔,和各种同等功效功能基团,显示的性质出与相应线型分子完全不同[18],如黏度低、溶解性好等,这些特性使其具有广泛的应用前景,近年来也成聚合物领域的研究热点,已有很多将其应用到蛋白质组学研究中的例子。论文的第一章综述了糖蛋白的研究意义和研究内容。第二章为探索性实验,探索了商品化树枝状聚合物PAMAM是否可以应用于糖肽富集。研究提出了一种新型的糖肽富集试剂,将不同代数、含高密度氨基末端的树枝状聚合物固定到溴化氰活化的琼脂糖凝胶上用于糖肽的高效分离,并对富集条件进行优化；第三章我们自合成了一种超分支聚合物：超支化缩水甘油(HPG),并利用SEM、TGA、1H-NMR、IR等手段表征其结构和外部形态。成功合成HPG以后,我们将合成的聚合物固定于微米尺寸的氨基硅球上,最后用己二酸二酰肼对结合后的聚合物进行酰肼修饰。结合各种表征手段验证新型酰肼材料被成功合成。我们发现基于两种载体材料制备的酰肼材料都具有很高的富集效率,从鼠脑全蛋白质提取液中,PAMAM共鉴定到133个糖蛋白、204条非冗余糖肽；HPG材料共鉴定到308个糖蛋白、726条非冗余糖肽。上述结果显示,我们发展的高分枝聚合物新材料,可以有效用于富集低丰度糖蛋白/糖肽。
[Abstract]:Glycosylation of proteins is one of the most common post-translational modifications. It not only plays a key role in the stability, solubility, folding and functional execution of proteins, but also participates in the transport, localization and molecular recognition of proteins in cells. In addition, abnormal glycosylation has been associated with diseases such as cancer, and many glycoproteins can be used as clinical biomarkers and therapeutic targets. Therefore, the separation, detection and identification of glycoproteins and glycopeptides have become more and more important, which can help us to better understand biological processes and find potential diagnostic markers and therapeutic targets. The qualitative and quantitative analysis of glycopeptide by mass spectrometry has become an important research direction in protein posttranslational modification. Since glycopeptides only account for a small portion of all the hydrolyzed peptides [7], the mass spectrum response is easily inhibited by high abundance non-glycopeptide. In addition, the microscopic heterogeneity of sugar chains further reduces the relative amount of glycopeptides and makes them difficult to be detected by mass spectrometry. Therefore, it is very important for the specific enrichment of glycopeptide. Lectin [8] and antibody [9] are the two most important affinity enrichment methods, but they have the disadvantages of poor stability and high price. Molecular exclusion, hydrophilic chromatography and boric acid enrichment are also widely used in the enrichment of glycopeptide. However, all kinds of enrichment methods have some shortcomings, which make the research progress of glycoproteomics more backward than other post-translational modifications such as phosphorylation, ubiquitization, acetylation and so on. In recent years, solid phase extraction of hydrazide as a traditional enrichment method of N-glycoprotein / glycopeptide has attracted much attention. But it also has some inevitable shortcomings: the sample loss is large, the steps are cumbersome. In addition, the most commonly used commercial hydrazide resins are not only expensive, but also have limited ability to bind glycoprotein / glycopeptide in solution. Therefore, the method of N-glycoprotein / glycopeptide enrichment by hydrazide is still to be improved. Highly branched polymers, due to their unique structural properties, such as accurate size, internal cavity, and various functional groups of equal efficacy, exhibit properties completely different from the corresponding linear molecules [18], such as low viscosity, good solubility, etc., These properties have a wide application prospect and have become a hot spot in the field of polymer research in recent years. There are many examples of their application in proteomics research. The first chapter summarizes the significance and content of glycoprotein research. The second chapter is an exploratory experiment to explore whether the commercial dendritic polymer PAMAM can be used in glycopeptide enrichment. A new glycopeptide enrichment reagent was proposed in this paper. The dendritic polymers with different algebras containing high density amino ends were immobilized on the agarose gel activated by cyanide bromide for the efficient separation of glycopeptides and the enrichment conditions were optimized. In chapter 3, we synthesized a superbranched polymer: hyperbranched glycidyl HPGN, and characterized its structure and external morphology by means of SEMGA-1H-NMR-IR. After the successful synthesis of HPG, the synthesized polymer was immobilized on the micron size aminosilicon spheres, and finally the polymer was modified with hydrazine adipate. The novel hydrazide materials were successfully synthesized by various characterization methods. We found that the hydrazide materials based on the two kinds of carrier materials have high enrichment efficiency. A total of 133 glycopeptides (204 non-redundant glycopeptides) were identified from brain protein extracts. A total of 308 glycoproteins and 726 non-redundant glycopeptides were identified from HPG materials. These results indicate that our new high branched polymer materials can be effectively used to enrich low abundance glycoprotein / glycopeptide.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R917

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