磷酸化蛋白质组学定性与定量分析技术的研究及在肝细胞磷酸化蛋白质组学中的应用

发布时间：2018-02-22 19:54

本文关键词： 磷酸化蛋白质组 SCX IPG-IEF-TiO_2 ~(18)O标记肝细胞　出处：《中国人民解放军军事医学科学院》2007年博士论文　论文类型：学位论文

【摘要】： 蛋白质的磷酸化修饰在生命活动中具有重要的调控作用，因此也是目前蛋白质组中翻译后修饰研究的热点，系统地对生物样本中磷酸化蛋白质组的研究有助于阐明磷酸化介导的生理病理机制。但由于生物体内磷酸化蛋白质的含量及磷酸化位点的化学计量值常常很低，大规模分析磷酸化蛋白质在技术上还很具有挑战性。肝脏在机体生命活动中执行多种重要生理功能，其中许多功能是通过可逆的磷酸化修饰调节的，对肝细胞模型的磷酸化蛋白质组的研究有助于人们更深入地理解和认识肝脏的复杂功能以及蛋白质的磷酸化修饰在肝脏功能中的意义。本研究分别从定性分析与定量分析两个层面对肝细胞磷酸化蛋白质组进行了研究。第一部分以标准磷酸化蛋白及酵母蛋白质为样本，首先优化了富集磷酸肽的SCX分离技术路线，接着采用SCX—Q—TOF／SCX—LCQ互补的分析策略对人Chang肝细胞中磷酸化蛋白质进行了研究。共鉴定到559个磷酸化位点、409个磷酸化肽段和370个磷酸化蛋白，其中69％(255／370)的磷酸化蛋白和82％(271／327)的磷酸化位点是Swiss-Prot数据库中没有报道的。分析发现其中许多蛋白质在生物体中发挥着与肝脏的生理和病理密切相关的重要功能。该数据集是目前第一个人正常肝细胞磷酸化蛋白质组的报道，为正常肝细胞与癌细胞对照分析提供了依据。第二部分首先建立了~(18)O标记的磷酸化肽段定量分析技术，并对方法学进行了考察和优化。之后建立了IPG—IEF—TiO_2联合分离富集方法与高准确度高灵敏度的LTQ—FT液相色谱质谱联用的磷酸化研究策略，通过比较考察发现该策略不但能有效地对磷酸化肽段进行富集，而且与~(18)O标记定量技术兼容性良好，，该工作目前还没有相关文献报道。辅助编写了~(18)O标记定量分析配套软件MSOQ，实现了~(18)O标记定量技术数据处理的自动化及规范化。最后将上述建立的~(18)O-IPO-IEF-TiO_2-LTQ-FT技术路线用于HepG2／HepG2-HBx细胞模型的定性及定量磷酸化蛋白质组研究。共鉴定到1358个磷酸化位点，958个磷酸化肽段和895个磷酸化蛋白质(groups)。85％的磷酸化蛋白和90％以上的磷酸化位点是Swiss-Prot数据库中没有报道的。从Chang细胞和HepG2细胞中都发现N端磷酸化肽段占很大比例的现象，推断肝细胞中磷酸化修饰有可能更容易发生在蛋白柔韧性好并暴露在外面的N端区域。稳定同位素标记定量分析得到三方面的差异定量信息：157个差异磷酸化位点，182个差异磷酸化蛋白质和1362个差异非磷酸化蛋白质。还发现有一些磷酸化蛋白质在蛋白水平与磷酸化位点的差异不一致的现象。以上信息的获得为探索由于HBx基因的整合而致HCC的分子机制，寻找一批与肝细胞癌发生发展紧密相关的高特异性和灵敏性的生物标记物奠定了基础。
[Abstract]:The phosphorylation modification of proteins plays an important role in the regulation of life activities, so it is also a hot topic in the research of post-translational modification in proteome. Systematic study of phosphorylated proteomics in biological samples helps to clarify the physiological and pathological mechanisms mediated by phosphorylation. However, the content of phosphorylated proteins and the stoichiometric values of phosphorylated sites in organisms are often very low. The large-scale analysis of phosphorylated proteins is also technically challenging. The liver performs a variety of important physiological functions in the life of the body, many of which are regulated by reversible phosphorylation modification. The study of phosphorylated proteome in hepatocyte model is helpful to understand the complex function of liver and the significance of phosphorylation modification of protein in liver function. In this study, the phosphorylated proteome of hepatocytes was studied from two aspects: qualitative analysis and quantitative analysis. In the first part, the standard phosphorylated protein and yeast protein were used as samples, and the SCX separation technique with rich phosphopeptide was optimized. Then the phosphorylated proteins in human Chang hepatocytes were studied by SCX-Q-TOF/SCX-LCQ complementary analysis. A total of #number0# phosphorylated peptides and 370 phosphorylated proteins were identified. The phosphorylation sites of the phosphorylated protein and the phosphorylation site of 822 / 271 / 327 are not reported in the Swiss-Prot database. Analysis shows that many of these proteins play an important role in the physiology and pathology of the liver in the organism. This data shows that the phosphorylation sites of the phosphorylated protein are closely related to the physiology and pathology of the liver. Set is the first report of phosphorylated proteome in normal human hepatocytes. It provides a basis for comparative analysis of normal hepatocytes and cancer cells. In the second part, a quantitative analysis technique of phosphorylated peptide segment labeled with 18 O was established. The methodology was investigated and optimized. Then the phosphorylation strategy of IPG-IEF-TiO_2 combined separation and enrichment method and high accuracy and high sensitivity LTQ-FT liquid chromatography-mass spectrometry (LC-MS) was established. It was found that the strategy could not only effectively enrich the phosphorylated peptide segment, but also had good compatibility with the quantitative technique for the detection of the phosphorylated peptide. At present, this work has not been reported in relevant literature. The software MSOQ has been compiled to realize the automation and standardization of the data processing of the quantitative analysis technique. Finally, the above technical route of 18O-IPO-IEF-TiO2-LTQ-FT has been used in the process of quantitative analysis. Qualitative and quantitative phosphorylation proteomics of HepG2/HepG2-HBx cell models. A total of 1 358 phosphorylation sites were identified, including 958 phosphorylated peptides and 895 phosphorylated proteins groups.85%, and more than 90% phosphorylation sites were identified as Swiss-Prot databases. A large proportion of N-terminal phosphorylated peptides were found in both Chang cells and HepG2 cells. It is inferred that phosphorylation modification in hepatocytes is more likely to occur in the N-terminal region where protein is flexible and exposed to the outside. Quantitative analysis of stable isotope labeling shows three different quantitative information: 157 differential phosphorylation sites. , 182 differentially phosphorylated proteins and 1362 differential non-phosphorylated proteins. There were also some differences between phosphorylated proteins at protein level and phosphorylation sites. The molecular mechanism of HCC due to its integration, Finding a batch of highly specific and sensitive biomarkers closely related to the occurrence and development of hepatocellular carcinoma has laid the foundation.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341

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