糖尿病肾病糖基化蛋白质组学及新型糖基化蛋白质富集材料的研究

发布时间：2018-04-02 13:39

本文选题：糖尿病肾病　切入点：糖基化蛋白质组学　出处：《新疆大学》2014年硕士论文

【摘要】：蛋白质糖基化是生命体内最为重要的翻译后修饰之一,参与了免疫应答、细胞间信号传导、细胞凋亡等几乎所有重要的生命过程,与众多重大疾病,如癌症的发生发展密切相关。研究表明很多疾病的诊断标志物和治疗靶标都是糖基化蛋白质,糖基化蛋白质组学研究在生命科学和临床医学上具有十分重要的意义。本论文以糖基化蛋白质为主要研究对象,开展了糖尿病肾病差异糖基化蛋白质组学研究,并针对糖基化蛋白质相当丰度较低、动态范围宽和质谱检测时易受非糖基化蛋白质干扰等问题,制备了两种新型糖基化蛋白质富集材料,主要研究内容如下： (1)通过“散弹枪”蛋白质组学策略对来自56例糖尿病肾病(包括：早期(亚临床期)、中期(临床期)和晚期(终末期))患者和19例对照组(健康组)的尿液进行研究。采用酰肼化学法富集N-糖基化肽段,HPLC-MS/MS对N-糖基化蛋白质进行分析和鉴定,采用基于Spectral counts的无标记方法对鉴定的糖基化蛋白质进行定量。共鉴定到295个糖基化蛋白质,其中25个异常表达。采用免疫印迹的方法对实验结果进行验证,结果证明数据可靠。经与文献对比,在25个异常表达的糖基化蛋白中,20个已被报道与糖尿病肾病或糖尿病、肾病相关,5个为首次发现与糖尿病肾病相关。 (2)亲水作用色谱法能富集各种类型的糖基化蛋白质/肽段,操作简单方便,不破坏糖链结构；磁性纳米材料是一类非常重要的无机功能材料,拥有具有巨大的比表面积、优异的磁响应能力和可修饰的表面。亲水磁性纳米材料将亲水作用色谱法和磁性纳米材料的优点结合起来,可以实现对糖肽的快速和高效的富集。传统的磁性亲水纳米材料的合成至少需要经历四个步骤,制备过程繁琐且耗时,本研究采用“一锅法”只需一步成功制备了壳聚糖包覆的磁性胶质纳米晶簇(Fe3O4@CS MCNCs)。并采用TG、SEM、Zeta电势、元素分析和磁滞曲线分析对其进行表征。该材料同时具有由晶簇表面的壳聚糖提供的强亲水性和传统磁性微球的特性,将之应用于标准糖基化蛋白质酶解物中糖肽的富集,表现出高选择性、低灵敏度(8fino)、高富集容量(35mg/g)以及快速分离等优良性能。将其应用于复杂样品Hela细胞,从45μg蛋白提取液共鉴定出175个糖基化蛋白(283个糖肽)。与传统四步法相比,该方法操作简单,经济。 (3)利用MIL-53家族具有的高比表面积、多孔结构和亲水特性,尝试将有机金属骨架(MOFs)应用于糖肽的选择性富集。以IgG酶解物为模型对MIL-53家族进行筛选,其中MIL-53(Al表现出对糖肽富集的高特异性、高灵敏度和高回收率。并对其富集机理进行探讨,.研究表明富集机理可能为亲水作用和尺寸排阻的双重作用效果。该发现为实现糖基化蛋白的大规模分析提供了可能,同时也拓宽了MOFs的应用领域。
[Abstract]:Glycosylation of proteins is one of the most important posttranslational modifications in life. It participates in immune response, intercellular signal transduction, apoptosis and so on. Studies have shown that many diagnostic markers and therapeutic targets are glycosylated proteins. Glycosylated proteomics is of great significance in life sciences and clinical medicine. In this paper, the differential glycosylation proteomics of diabetic nephropathy was studied. Two new glycosylated protein enrichment materials were prepared to solve the problems of relatively low glycosylated protein abundance, wide dynamic range and easy to be interfered by non-glycosylated protein in mass spectrometry. The main research contents are as follows:. Urine samples from 56 patients with diabetic nephropathy (including early (subclinical), intermediate (clinical) and late (end-stage)) and 19 controls (healthy group) were treated with "shotgun" proteomics strategy. The analysis and identification of N-glycosylated protein were carried out by HPLC-MS / MS enrichment of N-glycosylated peptide by hydrazide chemical method. A method based on Spectral counts was used to quantify the glycosylated proteins. A total of 295 glycosylated proteins were identified, 25 of which were expressed abnormally. The results were verified by Western blot. The results showed that the data were reliable. Of the 25 abnormal expression glycosylated proteins, 20 had been reported to be related to diabetic nephropathy or diabetic nephropathy, and 5 were first found to be related to diabetic nephropathy. Hydrophilic interaction chromatography can enrich all kinds of glycosylated protein / peptide segments, which is easy to operate and does not destroy the structure of sugar chain. Magnetic nanomaterials are a kind of very important inorganic functional materials with huge specific surface area. Excellent magnetic response and modifiable surfaces. Hydrophilic magnetic nanomaterials combine the advantages of hydrophilic interaction chromatography with magnetic nanomaterials, The synthesis of traditional magnetic hydrophilic nanomaterials requires at least four steps, and the preparation process is cumbersome and time-consuming. In this study, the magnetic colloid nanocrystalline Fe _ 3O _ 4CS MCNCsN coated with chitosan was successfully prepared by "one-pot method" and the Zeta potential was used. The material was characterized by elemental analysis and hysteresis curve analysis. The material has the properties of strong hydrophilicity and traditional magnetic microspheres provided by chitosan on the surface of the crystal cluster. It has been applied to the enrichment of glycopeptide in standard glycosylated protein hydrolysates. It showed excellent properties such as high selectivity, low sensitivity, high concentration capacity of 35 mg / g) and rapid separation. 175 glycosylated proteins were identified from 45 渭 g protein extracts from 45 渭 g protein extracts, and compared with the traditional four-step method, and compared with the traditional four-step method, 175 glycosylated proteins were identified from 45 渭 g protein extracts. The method is simple and economical. Using the high specific surface area, porous structure and hydrophilic properties of the MIL-53 family, we try to apply the organometallic skeleton mofs to selective enrichment of glycopeptide. The MIL-53 family was screened by using IgG hydrolysate as a model. Among them, MIL-53(Al showed high specificity for glycopeptide enrichment. The study shows that the enrichment mechanism may be the dual effect of hydrophilicity and size exclusion. This discovery provides the possibility for the large-scale analysis of glycosylated proteins. At the same time, it also widens the application field of MOFs.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q51;R587.2;R692.9

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