【摘要】：蛋白質(zhì)糖基化是一種結(jié)構(gòu)復(fù)雜、功能多樣的修飾類(lèi)型,糖基化修飾對(duì)蛋白質(zhì)的功能有重要影響,與許多疾病的發(fā)生、發(fā)展有著密切的關(guān)系,位點(diǎn)特異性的糖鏈結(jié)構(gòu)解析對(duì)揭示糖蛋白的生物學(xué)功能有重要意義。常規(guī)的糖蛋白質(zhì)組學(xué)研究方法,常利用內(nèi)切糖苷酶PNGase F切除糖鏈后,再鑒定糖修飾肽段及修飾位點(diǎn)。但糖鏈的切除,導(dǎo)致了糖型不均一性結(jié)構(gòu)的缺失,同時(shí),自發(fā)脫氨基現(xiàn)象會(huì)導(dǎo)致糖修飾位點(diǎn)鑒定的假陽(yáng)性結(jié)果。因此,發(fā)展能在完整糖肽水平上實(shí)現(xiàn)規(guī)�；⒏邷�(zhǔn)確性的糖肽結(jié)構(gòu)解析技術(shù),是糖蛋白質(zhì)組學(xué)研究需重點(diǎn)解決的技術(shù)難題,也是本研究所關(guān)注的核心內(nèi)容。本論文主要內(nèi)容分為三個(gè)章節(jié)。第一章,首先介紹了糖基化修飾對(duì)蛋白質(zhì)功能的影響和糖基化修飾的結(jié)構(gòu)特點(diǎn),其次介紹了常用的糖肽分離富集策略和生物質(zhì)譜檢測(cè)方法,同時(shí)還介紹了蛋白質(zhì)核心巖藻糖修飾在臨床研究與蛋白質(zhì)功能調(diào)控中的意義。第二章,建立了序列式內(nèi)切酶輔助的糖鏈結(jié)構(gòu)解析策略。在這項(xiàng)工作中,我們建立了一種位點(diǎn)特異性的糖鏈結(jié)構(gòu)解析策略,可用于實(shí)現(xiàn)復(fù)雜蛋白質(zhì)組樣品中完整N-糖肽的高可靠性鑒定。該策略首先是使用ZIC-HILIC富集蛋白酶解物中的完整糖肽。其次,將糖肽的一部分使用內(nèi)切糖苷酶H(Endo H)處理以去除高甘露糖型(Man)和雜合型N-連接的糖鏈。接著再將一部分Endo H處理后的糖肽進(jìn)一步在18O水中進(jìn)行PNGase F處理以除去剩余的復(fù)合型糖鏈。通過(guò)RPLCMS/MS分析三步處理所產(chǎn)生的樣本,包括完整的糖肽、Endo H處理糖肽、PNGase F處理糖肽,并且通過(guò)使用Byonic和p Find工具鑒定糖鏈結(jié)構(gòu)和肽段氨基酸序列。由于Endo H特異性識(shí)別高甘露糖和雜合糖結(jié)構(gòu),而PNGase F則可以切除Endo H消化后剩余的復(fù)合糖結(jié)果。通過(guò)第二步和第三步內(nèi)切糖苷酶的連續(xù)消化,所分別獲得的糖肽氨基酸序列信息可以提示糖肽所攜帶的糖鏈的類(lèi)型,進(jìn)而輔助提高第一步分析中所鑒定到的完整糖肽結(jié)構(gòu)的可靠性和鑒定準(zhǔn)確度。我們通過(guò)使用核糖核酸酶B(高甘露糖型)和IgG(復(fù)合糖結(jié)構(gòu)為主)驗(yàn)證了這種方法的有效性,并應(yīng)用這種順序消化策略實(shí)現(xiàn)HepG2細(xì)胞全蛋白中N-糖肽的分析。我們從HepG2細(xì)胞中成功鑒定947個(gè)特異糖修飾肽段的序列、1011個(gè)糖修飾位點(diǎn)、4514個(gè)位點(diǎn)特異性的糖型。同時(shí)獲得特定糖基化肽段上不同糖型的相對(duì)譜峰強(qiáng)度,實(shí)現(xiàn)了對(duì)位點(diǎn)特異性糖型占有率的分析。上述結(jié)果表明我們的方法可用于復(fù)雜樣品中的糖蛋白位點(diǎn)特異性的糖鏈結(jié)構(gòu)鑒定與定量分析。第三章,針對(duì)在腫瘤發(fā)生過(guò)程中具有重要標(biāo)志物價(jià)值的核心巖藻糖修飾,基于前期工作基礎(chǔ),開(kāi)展了肝癌、肝硬化、正常人血清核心巖藻糖化修飾肽段的大規(guī)模鑒定分析。對(duì)蛋白酶切之后得到的肽段,首先使用HILIC進(jìn)行糖肽的富集,然后使用LCH凝集素從得到的糖肽中富集核心巖藻糖修飾的肽段,再使用Endo F3酶切簡(jiǎn)化糖修飾肽段,最后通過(guò)LC-MS/MS檢測(cè),利用pFind搜庫(kù),可以獲得核心巖藻糖修飾的肽段及其質(zhì)譜響應(yīng)強(qiáng)度信息。在本研究中,首先將此方法應(yīng)用于C57小鼠肝臟和血清樣本中蛋白質(zhì)的核心巖藻糖修飾肽段的測(cè)試鑒定,實(shí)驗(yàn)結(jié)果表明富集的重現(xiàn)性良好,進(jìn)一步從正常人血清,肝硬化患者血清和肝癌患者血清中共鑒定到405個(gè)發(fā)生核心巖藻糖修飾的蛋白和879個(gè)肽段�；谫|(zhì)譜響應(yīng)強(qiáng)度的差異分析,驗(yàn)證了AFP的核心巖藻糖化在肝癌血清的高表達(dá)。發(fā)現(xiàn)GP73、PON1等蛋白質(zhì)的核心巖藻糖修飾在肝硬化或肝癌條件下的差異表達(dá),從而為新的肝病候選血清標(biāo)志物的發(fā)現(xiàn)提供線(xiàn)索。
[Abstract]:The glycosylation of the protein is a kind of modification type with complex structure and various functions, the glycosylation modification has an important influence on the function of the protein, and has a close relationship with the occurrence and development of many diseases, The analysis of the structure of the site-specific sugar chain is of great significance to reveal the biological function of the glycoprotein. In the conventional proteomics research, the sugar-modified peptide fragment and the modified site were identified by using the endoglucanase PNGase F to cut the sugar chain. However, the excision of the sugar chain leads to the deletion of the sugar-type non-uniformity structure, and at the same time, the spontaneous deamination phenomenon can lead to the false positive result of the identification of the sugar modification site. Therefore, the development of glycopeptide structure analysis technology which can realize the large-scale and high accuracy on the whole glycopeptide level is the technical problem that the research of the sugar proteomics needs to be focused on, and is the core content of the research of the research institute. The main contents of this thesis are divided into three chapters. In the first chapter, the effect of the glycosylation modification on the function of the protein and the structural characteristics of the glycosylation modification are introduced, and then the common glycopeptide separation and enrichment strategy and the biological mass spectrum detection method are introduced. The significance of protein core fucose modification in clinical research and protein function regulation was also introduced. In the second chapter, the sugar chain structure analysis strategy with the aid of the sequence endoglucanase is established. In this work, we have established a kind of site-specific sugar chain structure analysis strategy, which can be used for high-reliability identification of complete N-glycopeptides in complex protein group samples. The strategy is first to enrich the intact glycopeptides in the proteolytic enzyme using the ZIC-HILIC. Next, a portion of the glycopeptide is treated with an endoglucanase H (Endo H) to remove the high mannose type (Man) and the hybrid N-linked sugar chain. A portion of the Endo H-treated glycopeptide is then further treated with a PNGase F in 18O water to remove the remaining composite sugar chain. The samples produced by the three-step process of the RPLCMS/ MS analysis, including the complete glycopeptides, the Endo H-treated glycopeptides, the PNGase F-treated glycopeptides, and the identification of the sugar chain structure and the peptide-segment amino acid sequence by using the Byonic and p-Find tools. The Endo H specifically recognizes the high mannose and the hybrid sugar structure, while the PNGase F can cut the remaining complex sugar results after Endo H digestion. Through the continuous digestion of the second step and the third step endoglucanase, the obtained glycopeptide amino acid sequence information can be used for indicating the type of the sugar chain carried by the glycopeptide, and further improving the reliability and the identification accuracy of the whole glycopeptide structure identified in the first step analysis. The effectiveness of this method was verified by using ribonuclease B (high mannose type) and IgG (complex sugar structure), and the analysis of N-glycopeptides in HepG2 cells was achieved by this sequential digestion strategy. We successfully identified a sequence of 947 specific sugar-modified peptide segments,1011 sugar-modifying sites, and 4514-site-specific glycoforms from HepG2 cells. At the same time, the relative spectral peak intensity of the different glycoforms on the specific glycosylated peptide segment is obtained, and the analysis of the site-specific sugar-type occupancy rate is realized. The results show that the method can be used for the identification and quantitative analysis of the glycoprotein site-specific sugar chain structure in complex samples. In the third chapter, aiming at the core fucose modification with important marker value in the process of tumorigenesis, the large-scale identification and analysis of the core fucose modified peptide segment of the liver cancer, the liver cirrhosis and the normal human are carried out based on the preliminary work basis. The method comprises the following steps of: firstly, carrying out enrichment on the glycopeptide by using a HILIC, then enriching the core fucose-modified peptide segment from the obtained glycopeptide by using the LCH lectins, cutting the sugar-modified peptide segment by using the Endo F3 enzyme, and finally using the pFind search library through the LC-MS/ MS detection, The core fucose-modified peptide segment and its mass spectrum response strength information can be obtained. In this study, the method is first applied to the test and identification of the core fucose-modified peptide segment of the protein in the liver and the serum sample of the C57 mouse, There were 405 core fucose-modified proteins and 879 peptide fragments in serum and liver cancer patients in the patients with liver cirrhosis. The high expression of the core fucose of AFP in the serum of liver cancer was confirmed based on the differential analysis of the response intensity of mass spectrum. It was found that the core fucose of GP73, PON1 and other proteins was expressed in the condition of liver cirrhosis or liver cancer, thus providing a clue for the detection of the candidate serum markers of the new liver disease.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R91

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 梁軍;;復(fù)方天麻蜜環(huán)糖肽片治療椎基底動(dòng)脈供血不足效果觀(guān)察[J];河北醫(yī)藥;2008年06期

2 鄧北強(qiáng);周曉麗;;復(fù)方天麻蜜環(huán)糖肽片治療椎基底動(dòng)脈供血不足療效觀(guān)察[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2008年28期

3 曾麒燕,周德義,吳耀生,周素芳;紅桂木凝集素糖蛋白的特征及其糖肽鍵性質(zhì)的分析[J];廣西醫(yī)科大學(xué)學(xué)報(bào);1999年03期

4 張鏞;蘇偉展;張永雪;郝廣憲;;復(fù)方天麻蜜環(huán)糖肽片對(duì)腦卒中康復(fù)的臨床研究[J];中國(guó)臨床保健雜志;2007年06期

5 鄧珊珊;王明超;曹琦琛;白海紅;彭博;應(yīng)萬(wàn)濤;蔡耘;;超支化聚合物新材料富集糖肽方法的研究[J];中國(guó)生物工程雜志;2014年08期

6 楊衛(wèi)東，陳建茂，李紅兵，劉衛(wèi)忠，，馬明庭;生命神糖肽要素對(duì)動(dòng)物的影響[J];寧夏醫(yī)學(xué)院學(xué)報(bào);1994年03期

7 景洪江,薛長(zhǎng)勇;酸奶中糖肽復(fù)合物的分離純化[J];氨基酸和生物資源;2005年03期

8 張部昌,趙幟平,袁華玲,陳祖智,畢建軍;天門(mén)冬糖蛋白的分離純化及其糖肽鍵的研究[J];安徽大學(xué)學(xué)報(bào)(自然科學(xué)版);1996年04期

9 歐志福;;丹紅注射液聯(lián)合復(fù)方天麻蜜環(huán)糖肽片治療腦梗死[J];現(xiàn)代醫(yī)院;2010年08期

10 張群英;王成山;韓君勇;;復(fù)方天麻蜜環(huán)糖肽片治療女性更年期眩暈療效觀(guān)察[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2009年23期

相關(guān)會(huì)議論文 前5條

1 李艷梅;趙煒;陳永湘;蔡輝;趙鐳;劉沖;;具有生物活性的寡糖及糖肽的合成研究[A];中國(guó)化學(xué)會(huì)第26屆學(xué)術(shù)年會(huì)化學(xué)生物分會(huì)場(chǎng)論文集[C];2008年

2 梁鑫淼;曹麗偉;于龍;;糖肽的選擇性富集與酵母糖蛋白質(zhì)組分析[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第02分會(huì)：分離分析及微、納流控新方法[C];2014年

3 祁宏偉;楊華明;于維;劉海燕;;糖肽酮萜素在肉仔雞日糧中的應(yīng)用研究[A];動(dòng)物營(yíng)養(yǎng)與飼料研究——第五屆全國(guó)飼料營(yíng)養(yǎng)學(xué)術(shù)研討會(huì)論文集[C];2006年

4 龐蕾;謝建平;;糖肽脂對(duì)分枝桿菌生物膜的影響[A];2012年第五屆全國(guó)微生物遺傳學(xué)學(xué)術(shù)研討會(huì)論文摘要集[C];2012年

5 蔡輝;黃智華;石磊;孫占一;李艷梅;;用于腫瘤治療的糖肽疫苗的開(kāi)發(fā)[A];中國(guó)化學(xué)會(huì)第28屆學(xué)術(shù)年會(huì)第3分會(huì)場(chǎng)摘要集[C];2012年

相關(guān)博士學(xué)位論文 前1條

1 黃智華;自組裝MUC1糖肽腫瘤疫苗的設(shè)計(jì)、合成與應(yīng)用[D];清華大學(xué);2014年

相關(guān)碩士學(xué)位論文 前10條

1 牛陽(yáng)陽(yáng);守宮硫酸糖肽抑制肝癌細(xì)胞增殖與遷移的體外研究[D];天津醫(yī)科大學(xué);2016年

2 楊丹;度拉糖肽治療2型糖尿病的有效性和安全性系統(tǒng)評(píng)價(jià)[D];西南醫(yī)科大學(xué);2016年

3 于子翔;糖肽結(jié)構(gòu)解析與定量研究的組學(xué)技術(shù)發(fā)展與應(yīng)用[D];安徽醫(yī)科大學(xué);2017年

4 熊鵬;基于四肽聚合物的SiO_2色譜材料及其糖肽富集性能研究[D];武漢理工大學(xué);2015年

5 張懷遠(yuǎn);硼酸納米材料富集糖肽或糖蛋白[D];復(fù)旦大學(xué);2009年

6 鄧珊珊;基于高分枝聚合物材料富集糖肽新方法的研究[D];安徽醫(yī)科大學(xué);2014年

7 許林;云芝糖肽介導(dǎo)活化的人外周血單個(gè)核細(xì)胞體外抗乙肝病毒作用[D];南京中醫(yī)藥大學(xué);2010年

8 唐浩;復(fù)合納米材料用于糖肽和磷酸化肽選擇性富集質(zhì)譜鑒定新方法研究[D];復(fù)旦大學(xué);2012年

9 匡敏;基于功能化磁性納米材料的糖肽富集與質(zhì)譜鑒定新方法研究[D];復(fù)旦大學(xué);2013年

10 吳志剛;天然復(fù)雜型N-糖苷分離與糖肽的酶法合成[D];山東大學(xué);2009年

本文編號(hào)：2484532