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  本文關(guān)鍵詞： 磷酸化蛋白質(zhì)組 SCX IPG-IEF-TiO_2 ~(18)O標(biāo)記 肝細(xì)胞　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 蛋白質(zhì)的磷酸化修飾在生命活動中具有重要的調(diào)控作用，因此也是目前蛋白質(zhì)組中翻譯后修飾研究的熱點，系統(tǒng)地對生物樣本中磷酸化蛋白質(zhì)組的研究有助于闡明磷酸化介導(dǎo)的生理病理機(jī)制。但由于生物體內(nèi)磷酸化蛋白質(zhì)的含量及磷酸化位點的化學(xué)計量值常常很低，大規(guī)模分析磷酸化蛋白質(zhì)在技術(shù)上還很具有挑戰(zhàn)性。肝臟在機(jī)體生命活動中執(zhí)行多種重要生理功能，其中許多功能是通過可逆的磷酸化修飾調(diào)節(jié)的，對肝細(xì)胞模型的磷酸化蛋白質(zhì)組的研究有助于人們更深入地理解和認(rèn)識肝臟的復(fù)雜功能以及蛋白質(zhì)的磷酸化修飾在肝臟功能中的意義。 本研究分別從定性分析與定量分析兩個層面對肝細(xì)胞磷酸化蛋白質(zhì)組進(jìn)行了研究。 第一部分以標(biāo)準(zhǔn)磷酸化蛋白及酵母蛋白質(zhì)為樣本，首先優(yōu)化了富集磷酸肽的SCX分離技術(shù)路線，接著采用SCX—Q—TOF／SCX—LCQ互補(bǔ)的分析策略對人Chang肝細(xì)胞中磷酸化蛋白質(zhì)進(jìn)行了研究。共鑒定到559個磷酸化位點、409個磷酸化肽段和370個磷酸化蛋白，其中69％(255／370)的磷酸化蛋白和82％(271／327)的磷酸化位點是Swiss-Prot數(shù)據(jù)庫中沒有報道的。分析發(fā)現(xiàn)其中許多蛋白質(zhì)在生物體中發(fā)揮著與肝臟的生理和病理密切相關(guān)的重要功能。該數(shù)據(jù)集是目前第一個人正常肝細(xì)胞磷酸化蛋白質(zhì)組的報道，為正常肝細(xì)胞與癌細(xì)胞對照分析提供了依據(jù)。 第二部分首先建立了~(18)O標(biāo)記的磷酸化肽段定量分析技術(shù)，并對方法學(xué)進(jìn)行了考察和優(yōu)化。之后建立了IPG—IEF—TiO_2聯(lián)合分離富集方法與高準(zhǔn)確度高靈敏度的LTQ—FT液相色譜質(zhì)譜聯(lián)用的磷酸化研究策略，通過比較考察發(fā)現(xiàn)該策略不但能有效地對磷酸化肽段進(jìn)行富集，而且與~(18)O標(biāo)記定量技術(shù)兼容性良好，，該工作目前還沒有相關(guān)文獻(xiàn)報道。輔助編寫了~(18)O標(biāo)記定量分析配套軟件MSOQ，實現(xiàn)了~(18)O標(biāo)記定量技術(shù)數(shù)據(jù)處理的自動化及規(guī)范化。最后將上述建立的~(18)O-IPO-IEF-TiO_2-LTQ-FT技術(shù)路線用于HepG2／HepG2-HBx細(xì)胞模型的定性及定量磷酸化蛋白質(zhì)組研究。共鑒定到1358個磷酸化位點，958個磷酸化肽段和895個磷酸化蛋白質(zhì)(groups)。85％的磷酸化蛋白和90％以上的磷酸化位點是Swiss-Prot數(shù)據(jù)庫中沒有報道的。從Chang細(xì)胞和HepG2細(xì)胞中都發(fā)現(xiàn)N端磷酸化肽段占很大比例的現(xiàn)象，推斷肝細(xì)胞中磷酸化修飾有可能更容易發(fā)生在蛋白柔韌性好并暴露在外面的N端區(qū)域。穩(wěn)定同位素標(biāo)記定量分析得到三方面的差異定量信息：157個差異磷酸化位點，182個差異磷酸化蛋白質(zhì)和1362個差異非磷酸化蛋白質(zhì)。還發(fā)現(xiàn)有一些磷酸化蛋白質(zhì)在蛋白水平與磷酸化位點的差異不一致的現(xiàn)象。以上信息的獲得為探索由于HBx基因的整合而致HCC的分子機(jī)制，尋找一批與肝細(xì)胞癌發(fā)生發(fā)展緊密相關(guān)的高特異性和靈敏性的生物標(biāo)記物奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R341

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