AACT技术用于QSG-7701/QGY-7703定量蛋白质组研究并寻找肝癌发生相关分子标志物
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摘要
本论文主要通过蛋白质相对定量展开和深入研究与肝癌相关的工作。该论文运用的核心技术,氨基酸质量标签(amino acid coded-mass tagging,AACT)技术,是本课题组发明并发展的生物代谢标记结合质谱技术进行蛋白质准确定量的技术路线。研究对象是我国的重大疾病肝癌,在蛋白质水平系统研究其发生和转移在理论上为疾病机理探究、临床中为预诊和治疗的分子标志物的筛选都有重大意义。
本论文分为六章,主要工作集中在四部分:第一部分为氨基酸季铵化技术的初探;第二部分AACT定量蛋白质组技术平台的建立和优化;第三部分AACT技术平台用于对肝癌发生/转移相关的细胞株的定量蛋白组学的研究;最后亚细胞器蛋白质组研究中常规技术的优化工作。下面按照章节做介绍:
(1)前言部分对定量白质组技术的发展做了总结回顾,比较了各种技术的优缺点。着强调了“量”的概念,根据定量的准确性作了方法和技术的分类阐述。同时对肝癌蛋白质组学在国际上的研究水平和国内的现状作了总结,提出本论文选题的意义和创新点。
(2)氨基酸季铵化技术初探和定量蛋白质组学中的潜在应用:初步尝试用化学的方法解决实验过程中遇到的问题,发展了新的季铵化修饰的方法。该技术不仅大大提高了氨基酸/肽的离子化效率,而且可以在季铵化的过程中可以引入同位素进行定量标记。这一新方法在化学标记定量蛋白质组技术的舞台上有着广阔的应用前景。
(3)以生物质谱为基础的AACT蛋白质定量技术平台的建立:本论文将AACT技术运用到本课题组,建立并优化了整套稳定可靠的AACT定量蛋白质组技术平台。内容包括细胞株标记,同位素氨基酸的选择、标记程度的鉴定标准建立,LC-MS分离鉴定参数的优化,手动、自动定量标准的制定、标记技术的误差考察、AACT技术差异蛋白阈值的设定等,为本课题以及本课题组后续运用该技术定量蛋白质以及发展用于相互作用研究工作的开展奠定了坚实的理论和实验的基础。
(4)AACT技术用于QSG-7701/QGY-7703定量蛋白质组研究肝癌的发生:首先,研究对象选择一对典型的细胞模型,宿主肝细胞系QSG-7701和病灶部位肝癌细胞QGY-7703,这对细胞源自同一病人手术标本,是一对典型的细胞株用来研究原发性肝癌发生相关蛋白质组的模型。运用AACT技术首次在蛋水平研究QSG-7701/QGY-7703细胞株,提供了翔实的蛋白质表达谱和定量谱的数据。鉴定到1343个非冗余蛋白,511个蛋白有准确的定量信息,找到132个肝癌发生相关的蛋白质,有部分已经有文献报道,我们在肝癌中也找到同样的蛋白,有些是验证了已有和肝癌相关的,还有部分新发现与肝癌相关,western验证了部分蛋白质。在这一章节我们还对差异表达得的功能蛋白做了详尽讨论。通过进一步在组织中验证,提出了几个潜在的肝癌发生的标志物分子。
(5)AACT技术用于MHCC97H/L定量蛋白质组研究肝癌的转移:肝癌不能治愈主要原因来自其侵袭性即容易发生转移。复旦大学肝癌研究所建立了一对典型研究和肝癌转移潜能相关细胞株MHCC97H/L。我们进一步运用AACT技术的研究得到准确蛋白质定量谱,得到疾病转移相关蛋白定性和定量信息。深入了对疾病发生和发展的研究,提供了更加系统翔实的数据,为临床提供了诊断和药靶的候选分子,找到的差异蛋白可以帮助深入理解肝癌的发生发展机理。
(6)蛋白质组研究中常规技术的优化:针对细胞、小鼠肝脏和正常人肝样品优化了亚细胞器(细胞核和线粒体)的分离和纯化技术形成一整套方案。同时通过染色、电镜以及免疫组化技术对纯度作了系统评价。
The thesis was focused on quantitative proteomics platform of mass spectrometry based amino acid-coded tagging(AACT) technique.To investigate candidates of diagnostic or therapeutic biomarkers for liver cancers,we comprehensively measured systematic changes in protein expression of human hepatocellular carcinoma(HCC). The effective treatment of liver cancer relies on the diagnosis of the disease at an early stage.
The research work of this dissertation was composed of four parts.The first parts was about A novel method of quaternizing amino acid/peptide and its product in situ analysis using ESI-MS;In the second part,we set up a high throughput platform for global quantitative exploration of proteome using AACT proteomics technologies; The third part was comparative proteomic analysis of two paired HCC cell lines with the same genetic background to investigate markers for HCC and metastasis.And the last part was optimization routine technology for organelles.The details were described as the chapters in the following:
(1) The preface part summarized recent research progresses on quantitative proteomics and in the context of their respective strengths/weakness,especially emphasized the application of major amino acid-coded mass tagging based quantitative proteomics approaches.
(2) A new method of quaternizing amino acid and peptide was reported.This method could offer a potential chance to combine quanternizing and mass spectrometry which is helpful for the future use of quanternizing reaction in quantitative proteomics.
(3) We set up a high throughput platform for global exploration of quantitative proteome based on the amino acid coded-mass tagging technologies.Content includes cell labeling,choice of isotope amino acid,the label degree detection,LC-MS separates the optimization of parameter's,threshold value setting for quantification, quantify standard build up et.al..Finally we set up a platform for the following work in quantification and protein-protein interaction.
(4) Comparative proteomic analysis of a paired human liver normal-carcinoma cell lines with the same genetic background to investigate hepatocellular carcinoma Markers.Quantitative proteomic analysis of two cell lines derived from healthy liver and carcinoma tissue of a same donor,i.e.,QSG-7701 for the normal and QGY-7703 for the cancer cells respectively,was conducted using AACT.Among a total of 1343 proteins,511 was precisely quantified in HCC cells.According to their previously characterized functions,the differentially expressed proteins were found associated with five major functional categories.The accuracy of the AACT-based quantification was validated by Western blotting.Furthermore,the differentially expressed proteins were identified in HCC specimen.The consistency between cell lines and clinic tissues,comprised a part of reported biomarker for HCC.Altogether our study provides some insights into the molecular events that may lead to diverse strategies for diagnosis and treatment of HCC.
(5) Comprehensive profiling of metastasis-related proteins in paired hepatocelluar carcinoma cells with different metastasis potential.Metastasis is the primary cause of death in hepatocelluar carcinoma(HCC) patients.To identify those proteins associated with metastasis potential,we have conducted comparative proteomic analysis on a pair of the HCC cell lines characterized with different metastasis potential originated from a same donor(MHCC97L versus MHCC97H) by employing an amino acid-coded mass tagging(AACT)-based quantitative proteomic method.
(6) We optimized routine technology for organelle,especially for nucleus and mitochondrial in three level cells,mice tissues and healthy human liver.
本论文分为六章,主要工作集中在四部分:第一部分为氨基酸季铵化技术的初探;第二部分AACT定量蛋白质组技术平台的建立和优化;第三部分AACT技术平台用于对肝癌发生/转移相关的细胞株的定量蛋白组学的研究;最后亚细胞器蛋白质组研究中常规技术的优化工作。下面按照章节做介绍:
(1)前言部分对定量白质组技术的发展做了总结回顾,比较了各种技术的优缺点。着强调了“量”的概念,根据定量的准确性作了方法和技术的分类阐述。同时对肝癌蛋白质组学在国际上的研究水平和国内的现状作了总结,提出本论文选题的意义和创新点。
(2)氨基酸季铵化技术初探和定量蛋白质组学中的潜在应用:初步尝试用化学的方法解决实验过程中遇到的问题,发展了新的季铵化修饰的方法。该技术不仅大大提高了氨基酸/肽的离子化效率,而且可以在季铵化的过程中可以引入同位素进行定量标记。这一新方法在化学标记定量蛋白质组技术的舞台上有着广阔的应用前景。
(3)以生物质谱为基础的AACT蛋白质定量技术平台的建立:本论文将AACT技术运用到本课题组,建立并优化了整套稳定可靠的AACT定量蛋白质组技术平台。内容包括细胞株标记,同位素氨基酸的选择、标记程度的鉴定标准建立,LC-MS分离鉴定参数的优化,手动、自动定量标准的制定、标记技术的误差考察、AACT技术差异蛋白阈值的设定等,为本课题以及本课题组后续运用该技术定量蛋白质以及发展用于相互作用研究工作的开展奠定了坚实的理论和实验的基础。
(4)AACT技术用于QSG-7701/QGY-7703定量蛋白质组研究肝癌的发生:首先,研究对象选择一对典型的细胞模型,宿主肝细胞系QSG-7701和病灶部位肝癌细胞QGY-7703,这对细胞源自同一病人手术标本,是一对典型的细胞株用来研究原发性肝癌发生相关蛋白质组的模型。运用AACT技术首次在蛋水平研究QSG-7701/QGY-7703细胞株,提供了翔实的蛋白质表达谱和定量谱的数据。鉴定到1343个非冗余蛋白,511个蛋白有准确的定量信息,找到132个肝癌发生相关的蛋白质,有部分已经有文献报道,我们在肝癌中也找到同样的蛋白,有些是验证了已有和肝癌相关的,还有部分新发现与肝癌相关,western验证了部分蛋白质。在这一章节我们还对差异表达得的功能蛋白做了详尽讨论。通过进一步在组织中验证,提出了几个潜在的肝癌发生的标志物分子。
(5)AACT技术用于MHCC97H/L定量蛋白质组研究肝癌的转移:肝癌不能治愈主要原因来自其侵袭性即容易发生转移。复旦大学肝癌研究所建立了一对典型研究和肝癌转移潜能相关细胞株MHCC97H/L。我们进一步运用AACT技术的研究得到准确蛋白质定量谱,得到疾病转移相关蛋白定性和定量信息。深入了对疾病发生和发展的研究,提供了更加系统翔实的数据,为临床提供了诊断和药靶的候选分子,找到的差异蛋白可以帮助深入理解肝癌的发生发展机理。
(6)蛋白质组研究中常规技术的优化:针对细胞、小鼠肝脏和正常人肝样品优化了亚细胞器(细胞核和线粒体)的分离和纯化技术形成一整套方案。同时通过染色、电镜以及免疫组化技术对纯度作了系统评价。
The thesis was focused on quantitative proteomics platform of mass spectrometry based amino acid-coded tagging(AACT) technique.To investigate candidates of diagnostic or therapeutic biomarkers for liver cancers,we comprehensively measured systematic changes in protein expression of human hepatocellular carcinoma(HCC). The effective treatment of liver cancer relies on the diagnosis of the disease at an early stage.
The research work of this dissertation was composed of four parts.The first parts was about A novel method of quaternizing amino acid/peptide and its product in situ analysis using ESI-MS;In the second part,we set up a high throughput platform for global quantitative exploration of proteome using AACT proteomics technologies; The third part was comparative proteomic analysis of two paired HCC cell lines with the same genetic background to investigate markers for HCC and metastasis.And the last part was optimization routine technology for organelles.The details were described as the chapters in the following:
(1) The preface part summarized recent research progresses on quantitative proteomics and in the context of their respective strengths/weakness,especially emphasized the application of major amino acid-coded mass tagging based quantitative proteomics approaches.
(2) A new method of quaternizing amino acid and peptide was reported.This method could offer a potential chance to combine quanternizing and mass spectrometry which is helpful for the future use of quanternizing reaction in quantitative proteomics.
(3) We set up a high throughput platform for global exploration of quantitative proteome based on the amino acid coded-mass tagging technologies.Content includes cell labeling,choice of isotope amino acid,the label degree detection,LC-MS separates the optimization of parameter's,threshold value setting for quantification, quantify standard build up et.al..Finally we set up a platform for the following work in quantification and protein-protein interaction.
(4) Comparative proteomic analysis of a paired human liver normal-carcinoma cell lines with the same genetic background to investigate hepatocellular carcinoma Markers.Quantitative proteomic analysis of two cell lines derived from healthy liver and carcinoma tissue of a same donor,i.e.,QSG-7701 for the normal and QGY-7703 for the cancer cells respectively,was conducted using AACT.Among a total of 1343 proteins,511 was precisely quantified in HCC cells.According to their previously characterized functions,the differentially expressed proteins were found associated with five major functional categories.The accuracy of the AACT-based quantification was validated by Western blotting.Furthermore,the differentially expressed proteins were identified in HCC specimen.The consistency between cell lines and clinic tissues,comprised a part of reported biomarker for HCC.Altogether our study provides some insights into the molecular events that may lead to diverse strategies for diagnosis and treatment of HCC.
(5) Comprehensive profiling of metastasis-related proteins in paired hepatocelluar carcinoma cells with different metastasis potential.Metastasis is the primary cause of death in hepatocelluar carcinoma(HCC) patients.To identify those proteins associated with metastasis potential,we have conducted comparative proteomic analysis on a pair of the HCC cell lines characterized with different metastasis potential originated from a same donor(MHCC97L versus MHCC97H) by employing an amino acid-coded mass tagging(AACT)-based quantitative proteomic method.
(6) We optimized routine technology for organelle,especially for nucleus and mitochondrial in three level cells,mice tissues and healthy human liver.
引文
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