本文選題：糖尿病腎病　切入點：糖基化蛋白質組學　出處：《新疆大學》2014年碩士論文

【摘要】：蛋白質糖基化是生命體內最為重要的翻譯后修飾之一,參與了免疫應答、細胞間信號傳導、細胞凋亡等幾乎所有重要的生命過程,與眾多重大疾病,如癌癥的發(fā)生發(fā)展密切相關。研究表明很多疾病的診斷標志物和治療靶標都是糖基化蛋白質,糖基化蛋白質組學研究在生命科學和臨床醫(yī)學上具有十分重要的意義。本論文以糖基化蛋白質為主要研究對象,開展了糖尿病腎病差異糖基化蛋白質組學研究,并針對糖基化蛋白質相當豐度較低、動態(tài)范圍寬和質譜檢測時易受非糖基化蛋白質干擾等問題,制備了兩種新型糖基化蛋白質富集材料,主要研究內容如下： (1)通過“散彈槍”蛋白質組學策略對來自56例糖尿病腎病(包括：早期(亞臨床期)、中期(臨床期)和晚期(終末期))患者和19例對照組(健康組)的尿液進行研究。采用酰肼化學法富集N-糖基化肽段,HPLC-MS/MS對N-糖基化蛋白質進行分析和鑒定,采用基于Spectral counts的無標記方法對鑒定的糖基化蛋白質進行定量。共鑒定到295個糖基化蛋白質,其中25個異常表達。采用免疫印跡的方法對實驗結果進行驗證,結果證明數(shù)據(jù)可靠。經與文獻對比,在25個異常表達的糖基化蛋白中,20個已被報道與糖尿病腎病或糖尿病、腎病相關,5個為首次發(fā)現(xiàn)與糖尿病腎病相關。 (2)親水作用色譜法能富集各種類型的糖基化蛋白質/肽段,操作簡單方便,不破壞糖鏈結構；磁性納米材料是一類非常重要的無機功能材料,擁有具有巨大的比表面積、優(yōu)異的磁響應能力和可修飾的表面。親水磁性納米材料將親水作用色譜法和磁性納米材料的優(yōu)點結合起來,可以實現(xiàn)對糖肽的快速和高效的富集。傳統(tǒng)的磁性親水納米材料的合成至少需要經歷四個步驟,制備過程繁瑣且耗時,本研究采用“一鍋法”只需一步成功制備了殼聚糖包覆的磁性膠質納米晶簇(Fe3O4@CS MCNCs)。并采用TG、SEM、Zeta電勢、元素分析和磁滯曲線分析對其進行表征。該材料同時具有由晶簇表面的殼聚糖提供的強親水性和傳統(tǒng)磁性微球的特性,將之應用于標準糖基化蛋白質酶解物中糖肽的富集,表現(xiàn)出高選擇性、低靈敏度(8fino)、高富集容量(35mg/g)以及快速分離等優(yōu)良性能。將其應用于復雜樣品Hela細胞,從45μg蛋白提取液共鑒定出175個糖基化蛋白(283個糖肽)。與傳統(tǒng)四步法相比,該方法操作簡單,經濟。 (3)利用MIL-53家族具有的高比表面積、多孔結構和親水特性,嘗試將有機金屬骨架(MOFs)應用于糖肽的選擇性富集。以IgG酶解物為模型對MIL-53家族進行篩選,其中MIL-53(Al表現(xiàn)出對糖肽富集的高特異性、高靈敏度和高回收率。并對其富集機理進行探討,.研究表明富集機理可能為親水作用和尺寸排阻的雙重作用效果。該發(fā)現(xiàn)為實現(xiàn)糖基化蛋白的大規(guī)模分析提供了可能,同時也拓寬了MOFs的應用領域。
[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.
【學位授予單位】：新疆大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：Q51;R587.2;R692.9

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