糖肽结构解析与定量研究的组学技术发展与应用

发布时间：2019-05-24 03:45

【摘要】：蛋白质糖基化是一种结构复杂、功能多样的修饰类型,糖基化修饰对蛋白质的功能有重要影响,与许多疾病的发生、发展有着密切的关系,位点特异性的糖链结构解析对揭示糖蛋白的生物学功能有重要意义。常规的糖蛋白质组学研究方法,常利用内切糖苷酶PNGase F切除糖链后,再鉴定糖修饰肽段及修饰位点。但糖链的切除,导致了糖型不均一性结构的缺失,同时,自发脱氨基现象会导致糖修饰位点鉴定的假阳性结果。因此,发展能在完整糖肽水平上实现规模化、高准确性的糖肽结构解析技术,是糖蛋白质组学研究需重点解决的技术难题,也是本研究所关注的核心内容。本论文主要内容分为三个章节。第一章,首先介绍了糖基化修饰对蛋白质功能的影响和糖基化修饰的结构特点,其次介绍了常用的糖肽分离富集策略和生物质谱检测方法,同时还介绍了蛋白质核心岩藻糖修饰在临床研究与蛋白质功能调控中的意义。第二章,建立了序列式内切酶辅助的糖链结构解析策略。在这项工作中,我们建立了一种位点特异性的糖链结构解析策略,可用于实现复杂蛋白质组样品中完整N-糖肽的高可靠性鉴定。该策略首先是使用ZIC-HILIC富集蛋白酶解物中的完整糖肽。其次,将糖肽的一部分使用内切糖苷酶H(Endo H)处理以去除高甘露糖型(Man)和杂合型N-连接的糖链。接着再将一部分Endo H处理后的糖肽进一步在18O水中进行PNGase F处理以除去剩余的复合型糖链。通过RPLCMS/MS分析三步处理所产生的样本,包括完整的糖肽、Endo H处理糖肽、PNGase F处理糖肽,并且通过使用Byonic和p Find工具鉴定糖链结构和肽段氨基酸序列。由于Endo H特异性识别高甘露糖和杂合糖结构,而PNGase F则可以切除Endo H消化后剩余的复合糖结果。通过第二步和第三步内切糖苷酶的连续消化,所分别获得的糖肽氨基酸序列信息可以提示糖肽所携带的糖链的类型,进而辅助提高第一步分析中所鉴定到的完整糖肽结构的可靠性和鉴定准确度。我们通过使用核糖核酸酶B(高甘露糖型)和IgG(复合糖结构为主)验证了这种方法的有效性,并应用这种顺序消化策略实现HepG2细胞全蛋白中N-糖肽的分析。我们从HepG2细胞中成功鉴定947个特异糖修饰肽段的序列、1011个糖修饰位点、4514个位点特异性的糖型。同时获得特定糖基化肽段上不同糖型的相对谱峰强度,实现了对位点特异性糖型占有率的分析。上述结果表明我们的方法可用于复杂样品中的糖蛋白位点特异性的糖链结构鉴定与定量分析。第三章,针对在肿瘤发生过程中具有重要标志物价值的核心岩藻糖修饰,基于前期工作基础,开展了肝癌、肝硬化、正常人血清核心岩藻糖化修饰肽段的大规模鉴定分析。对蛋白酶切之后得到的肽段,首先使用HILIC进行糖肽的富集,然后使用LCH凝集素从得到的糖肽中富集核心岩藻糖修饰的肽段,再使用Endo F3酶切简化糖修饰肽段,最后通过LC-MS/MS检测,利用pFind搜库,可以获得核心岩藻糖修饰的肽段及其质谱响应强度信息。在本研究中,首先将此方法应用于C57小鼠肝脏和血清样本中蛋白质的核心岩藻糖修饰肽段的测试鉴定,实验结果表明富集的重现性良好,进一步从正常人血清,肝硬化患者血清和肝癌患者血清中共鉴定到405个发生核心岩藻糖修饰的蛋白和879个肽段。基于质谱响应强度的差异分析,验证了AFP的核心岩藻糖化在肝癌血清的高表达。发现GP73、PON1等蛋白质的核心岩藻糖修饰在肝硬化或肝癌条件下的差异表达,从而为新的肝病候选血清标志物的发现提供线索。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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