新型亲水富集材料的制备及正常人尿液糖基化蛋白的富集鉴定

发布时间：2018-05-29 21:52

本文选题：尿蛋白 + N-糖肽　；参考：《华北理工大学》2017年硕士论文

【摘要】：目的1蛋白质的N-糖基化修饰参与了许多重要的生物过程,是多种重大疾病诊断标志物研究的热点。与其他体液活检样本相比,尿液可以无创,大量获取,并且不受稳态调节的影响,可以在一定程度上反映整个机体的生理和病理状态。因此对人尿液蛋白质N-糖基化的规模化研究对疾病标记物的筛选和治疗靶点的发现均具有重大意义;2由于人尿液中的N-糖蛋白含量有限,修饰比例较低,在质谱分析时N-糖肽易被高丰度的非糖肽所掩盖,难于鉴定。因此,发展高效、高选择性的富集材料是实现尿蛋白N-糖基化深度覆盖的必要条件。方法1使用亲水填料富集30例健康人尿液中的蛋白酶解后的肽段,并将富集到的肽段进行液质联用质谱鉴定,把得到的质谱数据导入分析软件,获得糖肽及糖基化位点信息;2先在硅球表面接上巯基,然后利用巯基-烯点击化学方法,将带有烯基的两性离子亲水单体键合到硅球表面。结果1男女分别鉴定到946和880个糖蛋白,利用生物信息学软件GO进行分析得到男女尿蛋白信息;2使用[3-(甲基丙烯酰氨基)丙基]二甲基(3-硫代丙基)氢氧化铵内盐(SPP)制备了两性离子修饰亲水硅胶材料(SPP-Si O2),将材料应用于标准糖蛋白和健康人尿蛋白N-糖肽的富集和质谱检测,从Ig G中鉴定到36条肽段,三个批次不同的SPP-Si O2材料分别富集并质谱鉴定959个,837个和877个N-糖基化位点,选择性均超过70%,从人尿液中一共鉴定到包含1065个位点的633个尿液糖蛋白,比文献报道的要多12.2%。其中81.2%的蛋白和73.9%的位点在至少两次富集中得到鉴定。结论1成功建立了稳定、高效的人尿液糖型及糖基化蛋白质规模化定量分析策略,并初步获得大规模的健康人尿液蛋白质糖基化修饰数据集,为实现筛选潜在生物标记物打下基础;2成功合成新型两性离子亲水富集材料,并成功应用于复杂样本,明显的提高了对肽段的富集效率。
[Abstract]:Objective 1 the N-glycosylation modification of proteins participates in many important biological processes and is a hot spot in the research of many major disease diagnostic markers. Compared with other body fluid biopsy samples, urine can be obtained in a non-invasive manner, and not affected by steady-state regulation, which can reflect the physiological and pathological state of the whole body to some extent. Therefore, the study on the scale of N-glycosylation of human urine protein is of great significance to the screening of disease markers and the discovery of therapeutic targets. 2 because of the limited content of N-glycoprotein in human urine, the modified proportion is relatively low. N-glycopeptide is easily masked by high abundance non-glycopeptide in mass spectrometry, so it is difficult to identify. Therefore, the development of highly efficient and highly selective enriched materials is a necessary condition for the deep coverage of urine protein N-glycosylation. Methods 1Hydrophilic fillers were used to enrich the peptides in urine of 30 healthy people after proteolysis, and the peptides were identified by liquid-mass spectrometry, and the obtained mass spectrometry data were imported into the analysis software. The glycopeptide and glycosylation site information were obtained. The glycosylated amphiphilic monomer with alkenyl group was bonded to the surface of the silicon sphere by mercapto-ene click-chemical method. Results 1 946 and 880 glycoproteins were identified in men and women respectively. Using the bioinformatics software go to analyze the urinary protein information of men and women, the amphoteric modified hydrophilic silica material SPP-Si was prepared using [3 (methacrylaminyl) propyl] dimethylpropyl) ammonium hydroxide (SPP). The material was applied to the enrichment and mass spectrometric analysis of standard glycoprotein and urine protein N-glycopeptide in healthy people. 36 peptides were identified from Ig G, and three batches of SPP-Si O 2 were enriched and identified by mass spectrometry (MS), respectively, with 959 N-glycosylation sites and 877 N-glycosylation sites. The selectivity was more than 70. A total of 633 urine glycoproteins containing 1065 sites were identified from human urine, which is 12.2 more than reported in the literature. 81.2% of the proteins and 73.9% of the loci were identified in at least two enrichment processes. Conclusion 1 A stable and efficient quantitative analysis strategy for glycosylated protein and glycosylated protein in human urine was successfully established, and a large scale data set of glycosylation modification of human urine was obtained. The new amphoteric ion hydrophilic enrichment materials were successfully synthesized and applied to complex samples for screening potential biomarkers. The efficiency of peptide enrichment was improved obviously.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O652.6;R446.12

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