本文選題：高分枝聚合物 + 酰肼富集��； 參考：《安徽醫(yī)科大學(xué)》2014年碩士論文

【摘要】：蛋白質(zhì)糖基化作為一種最常見的翻譯后修飾。它不僅在蛋白質(zhì)的穩(wěn)定性、溶解性、折疊和功能執(zhí)行方面起著關(guān)鍵的作用,而且參與細胞內(nèi)蛋白質(zhì)的運輸、定位和分子識別。此外,異常糖基化也已經(jīng)被證明和癌癥等疾病相關(guān),許多糖蛋白可以作為臨床生物標志物和治療靶標。因此,糖蛋白和糖肽的分離、發(fā)現(xiàn)和鑒定變得越來越重要,它可以幫助我們更好地理解生物過程,尋找潛在的診斷標志物和治療靶點。依靠生物質(zhì)譜對糖肽規(guī)模化定性與定量分析,已成為當前蛋白質(zhì)翻譯后修飾研究的一個重要研究方向。由于糖肽僅占所有酶解后肽段的小部分(2%～5%)[7],其質(zhì)譜響應(yīng)很容易被高豐度非糖肽抑制。另外,糖鏈的微觀不均一性進一步降低了糖肽的相對量而使得它們難以被質(zhì)譜檢測。因此,對于糖肽的特異性富集至關(guān)重要。凝集素[8]和抗體[9]是最重要的兩種親和富集方法,但具有穩(wěn)定性差,價格昂貴的缺點。分子排阻、親水色譜、硼酸富集也都廣泛應(yīng)用于糖肽的富集。但是,各種富集手段都存在一定的缺點,這些都使得糖蛋白組學(xué)的研究進展相對于磷酸化、泛素化、乙�；绕渌g后修飾,更為落后。近年來,酰肼固相提取法作為傳統(tǒng)的富集N-糖蛋白/糖肽的富集方法也備受關(guān)注。但是它也存在一些不可避免的缺點：樣品損耗大,步驟繁瑣。此外,最常用的商業(yè)化酰肼樹脂不僅價格高,而且在溶液中結(jié)合糖蛋白/糖肽能力有限。因此,目前依靠酰肼富集N-糖蛋白/糖肽的方法還有待完善。 高分枝聚合物由于其獨特的結(jié)構(gòu)特性,如準確的大小,內(nèi)部空腔,和各種同等功效功能基團,顯示的性質(zhì)出與相應(yīng)線型分子完全不同[18],如黏度低、溶解性好等,這些特性使其具有廣泛的應(yīng)用前景,近年來也成聚合物領(lǐng)域的研究熱點,已有很多將其應(yīng)用到蛋白質(zhì)組學(xué)研究中的例子。 論文的第一章綜述了糖蛋白的研究意義和研究內(nèi)容。第二章為探索性實驗,探索了商品化樹枝狀聚合物PAMAM是否可以應(yīng)用于糖肽富集。研究提出了一種新型的糖肽富集試劑,將不同代數(shù)、含高密度氨基末端的樹枝狀聚合物固定到溴化氰活化的瓊脂糖凝膠上用于糖肽的高效分離,并對富集條件進行優(yōu)化；第三章我們自合成了一種超分支聚合物：超支化縮水甘油(HPG),并利用SEM、TGA、1H-NMR、IR等手段表征其結(jié)構(gòu)和外部形態(tài)。成功合成HPG以后,我們將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對結(jié)合后的聚合物進行酰肼修飾。結(jié)合各種表征手段驗證新型酰肼材料被成功合成。我們發(fā)現(xiàn)基于兩種載體材料制備的酰肼材料都具有很高的富集效率,從鼠腦全蛋白質(zhì)提取液中,PAMAM共鑒定到133個糖蛋白、204條非冗余糖肽；HPG材料共鑒定到308個糖蛋白、726條非冗余糖肽。上述結(jié)果顯示,我們發(fā)展的高分枝聚合物新材料,可以有效用于富集低豐度糖蛋白/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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R917

【參考文獻】

相關(guān)期刊論文 前1條

1 鄧珊珊;曹琦琛;馬成;白海紅;任曉君;應(yīng)萬濤;蔡耘;;一種基于聚酰胺-胺型樹枝狀聚合物富集糖肽的新策略[J];生物技術(shù)通訊;2014年02期

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本文編號：1839599