本文選題：尿蛋白 + N-糖肽　； 參考：《華北理工大學》2017年碩士論文

【摘要】：目的1蛋白質(zhì)的N-糖基化修飾參與了許多重要的生物過程,是多種重大疾病診斷標志物研究的熱點。與其他體液活檢樣本相比,尿液可以無創(chuàng),大量獲取,并且不受穩(wěn)態(tài)調(diào)節(jié)的影響,可以在一定程度上反映整個機體的生理和病理狀態(tài)。因此對人尿液蛋白質(zhì)N-糖基化的規(guī)�；芯繉膊擞浳锏暮Y選和治療靶點的發(fā)現(xiàn)均具有重大意義;2由于人尿液中的N-糖蛋白含量有限,修飾比例較低,在質(zhì)譜分析時N-糖肽易被高豐度的非糖肽所掩蓋,難于鑒定。因此,發(fā)展高效、高選擇性的富集材料是實現(xiàn)尿蛋白N-糖基化深度覆蓋的必要條件。方法1使用親水填料富集30例健康人尿液中的蛋白酶解后的肽段,并將富集到的肽段進行液質(zhì)聯(lián)用質(zhì)譜鑒定,把得到的質(zhì)譜數(shù)據(jù)導(dǎo)入分析軟件,獲得糖肽及糖基化位點信息;2先在硅球表面接上巰基,然后利用巰基-烯點擊化學方法,將帶有烯基的兩性離子親水單體鍵合到硅球表面。結(jié)果1男女分別鑒定到946和880個糖蛋白,利用生物信息學軟件GO進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)氫氧化銨內(nèi)鹽(SPP)制備了兩性離子修飾親水硅膠材料(SPP-Si O2),將材料應(yīng)用于標準糖蛋白和健康人尿蛋白N-糖肽的富集和質(zhì)譜檢測,從Ig G中鑒定到36條肽段,三個批次不同的SPP-Si O2材料分別富集并質(zhì)譜鑒定959個,837個和877個N-糖基化位點,選擇性均超過70%,從人尿液中一共鑒定到包含1065個位點的633個尿液糖蛋白,比文獻報道的要多12.2%。其中81.2%的蛋白和73.9%的位點在至少兩次富集中得到鑒定。結(jié)論1成功建立了穩(wěn)定、高效的人尿液糖型及糖基化蛋白質(zhì)規(guī)�；糠治霾呗�,并初步獲得大規(guī)模的健康人尿液蛋白質(zhì)糖基化修飾數(shù)據(jù)集,為實現(xiàn)篩選潛在生物標記物打下基礎(chǔ);2成功合成新型兩性離子親水富集材料,并成功應(yīng)用于復(fù)雜樣本,明顯的提高了對肽段的富集效率。
[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.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O652.6;R446.12

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