氨基酸同位素标记技术在肝癌疾病蛋白质组学研究中的应用
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摘要
肝癌是世界上发生最广的癌症之一,在我国也是一种常发生的恶性肿瘤。肝癌早期症状隐匿,不容易察觉,绝大部分患者一旦确诊往往已属于晚期,或者在手术后较短时间内转移到其他器官,导致死亡。探讨肝癌相关分子及分子机制,寻找肝癌的生物标记物和治疗的靶分子,对于恶性肿瘤的诊断和治疗具有重要的理论指导和临床应用意义。利用定量蛋白质组技术从全基因组水平来研究生物学现象,比较肝癌细胞系和肝细胞系以及比较具有不同转移潜能的肝癌细胞系的蛋白质组差异,将有助于对肝癌机理的理解,最终指导肝癌诊断和治疗。
本论文以氨基酸同位素标记技术(AACT/SILAC)为技术平台,首先发展了一种判断标记肽假阳性的方法,剔除假阳性标记肽对定量的影响,改进了氨基酸同位素标记技术。最后选择了具有相同遗传学背景的两对细胞系,研究了肝癌及转移过程中蛋白质组的差异,获得一批潜在的肝癌分子标志物。此外本论文初步研究了不同搜索软件对定量结果的影响,为定量蛋白质组研究选择搜索软件提供依据(见附录)。
本论文的第一部分综述了定量蛋白质组研究的发展情况,首先对质谱仪器做了介绍,然后介绍了各种定量方法,比较了各个方法的优缺点。
本论文的第二部分改进了氨基酸同位素标记技术,并研究了同肝癌及转移有关的差异蛋白质组,具体包括:
(一)标记肽假阳性的判断
本部分发展了一种判断氨基酸同位素标记方法中标记肽假阳性的方法,计算出不同标记效率下的理论同位素分布,利用最小二乘法拟合得到标记肽的标记效率,根据假阳性阈值来判断标记肽的假阳性。本方法经实际应用,证明简单易行可靠。
(二)高转移肝癌细胞系的表达谱
蛋白质表达谱是了解肝癌的分子基础,在本实验中,利用SDS-PAGE分离结合LC-MALDI-MS鉴定技术,获得了高转移肝癌细胞系MHCC97H的蛋白质表达谱,系统分析了MHCC97H的蛋白质表达谱特点。本部分研究首先分析了表达谱的分子量和pI等,显示了本方法在鉴定具有极端分子量和pI的蛋白质方面具有的优势,发现蛋白质的细胞定位和其pI之间有一定的关联,利用GeneOntology在三个方面对表达谱蛋白做了GO注释,并利用InterPro数据库分析了表达谱的蛋白质家族特性。根据KEGG的数据和HPRD中的蛋白质相互作用数据,分析了表达谱蛋白所涉及的KEGG通路和蛋白质相互作用。
(三)肝癌差异谱的研究
本部分试验比较了肝癌细胞系QGY-7703和肝细胞系QSG-7701的蛋白质组差异,鉴定到一批和肝癌有关的蛋白质分子,这些蛋白质分子在细胞凋亡、增殖和细胞移动中发挥着重要的作用,借助Western blotting和免疫组化验证了定量结果的准确性和在临床样本中差异蛋白的表现。同时本部分研究利用GeneOntology、KEGG、蛋白质相互作用分析了差异蛋白的生物学功能,发现多个差异蛋白参与糖酵解和三羧酸循环等能量代谢,差异蛋白在MAPK、钙离子通路、Wnt等信号通路中也发挥作用。此外讨论了热激蛋白、过氧化物还原蛋白、膜联蛋白、角蛋白等在肝癌中的生物学功能。
(四)肝癌转移差异谱的研究
本部分试验利用AACT/SILAC技术比较了具有高转移能力的肝癌细胞系MHCC97H和低转移能力的肝癌细胞系MHCC97L的蛋白质组差异。利用Western blotting技术,在5个具有不同转移能力的肝癌细胞系中,检验了差异蛋白和肝癌转移能力的关系,同时也比较了差异蛋白在肝癌临床样本和癌旁组织的表达差异。利用生物信息工具,分析了差异蛋白的GeneOntology、KEGG通路和蛋白质相互作用,并在细胞增殖和凋亡、细胞移动及血管生成等方面讨论了差异蛋白的生物学功能,揭示他们在肝癌转移过程的作用。
本论文改进了氨基酸同位素标记技术,利用AACT/SILAC技术全面系统的分析了肝癌及转移过程中的蛋白质组变化,在蛋白质水平研究了肝癌中发生的一系列分子事件,探讨了肝癌的分子机制,研究和发掘一批与肝癌密切相关的蛋白质分子,必将有利于深入、系统的理解肝癌机制,促进肝癌生物标志物的发掘和临床诊断应用。
Hepatocellular carcinoma (HCC) is known as a one of difficultly curable diseases in the world and in China. Surgery remains the primary treatment of choice, but only approximately 6% of patients survive more than five years after resection. The major obstacle for the prolonging survival of HCC patients is metastasis after resection. Meanwhile the molecular mechanism underlying the HCC still has yet to be completely understood. Efforts to assess proteins involved in HCC are critical to elucidate potential biomarkers and therapy targets in HCC. Quantitative proteomics or comparative proteomics aims to screen the proteome differences associated with disease phenotype, and discovers the proteins functioning in the process of HCC. Systematic understanding using quantitative proteomics by comparing the HCC cell line versus liver cell line or by comparing HCC cell lines with different metastasis potential will show light on the molecular mechanisms of HCC and provide potential biomarkers for early diagnosis and therapy.
The present works developed one method to determine the false positive of labeled peptide in amino acid-coded mass tagging/stable isotope labeling with amino acids in cultured cell (AACT/SILAC). The works also comprehensively profiled the differential protein expression pattern using AACT/SILAC in HCC, and provided many useful biomarker candidates for HCC.
Firstly the work provided a brief summary about the development of quantitative proteomics, introducing the MS instrument and quantification methods such as 2DE、ICAT、iTRAQ and AACT/SILAC.
Secondly the work improved the AACT/SILAC technology and focused on the differential proteome of HCC, and was illustrated as following.
(1) Evaluation of the false positive of labeled peptide in AACT/SILAC
Stable isotope labeling became the chief technology in quantitative proteomics and false positive of peptide labeled was not well studied yet for the present. This article developed a method to judge the false positive of labeled peptide. Based on the probability model of isotope distribution, theoretical isotope distribution with different label efficiency were calculated, the label efficiency of peptide labeled was evaluated by comparing the experimental and theoretical isotope distribution using the least-square method. The peptide was determined as a false-positive one when its label efficiency was below the threshold. This judgment method would facilitate the precisely quantification of AACT/SILAC in proteomics.
(2) Proteome expression profile of HCC cell line with high metastasis potential
Proteome expression profile was the basis to understand the HCC mechanism and therapy. Proteins identified in HCC cell lines with high metastasis potential were put into biological context with various annotation sources followed by a bioinformatics analysis. The profile was annotated in genomic context and characterized the correlation between subcellular location and the pI. Proteins were further annotated with GeneOntology, grouped into proteins families in InterPro database and mapped to KEGG pathways they were involved. Advantages of AACT/SILAC over 2DE in identifying the proteins with high molecular weight and pI or membrane proteins were demonstrated. The protein-protein interaction network derived from HPRD was also examined.
(3) Differential proteome profile between a HCC cell line and liver cell line
Proteome alteration between two cell lines derived from HCC tissue and its adjacent tissue were screened using AACT/SILAC and differential proteome profile containing 224 proteins were obtained. Western blotting and immune histochemistry also validated the accuracy of the quantification results and the expression alteration of representative proteins in clinical HCC tissues. Biological functions of differentially expressed proteins were annotated by GeneOntology, KEGG and protein-protein interaction. These proteins were mapped to MAPK, calcium, Wnt signal pathway which regulated the cell proliferation and cell migration and functioned in HCC. Functions of annexin family, heat shock proteins and cytokeratin family in HCC were also disscused.
(4) Differential proteome profile between HCC cell lines with different metastasis potential
Proteome profile of two HCC cell lines with high and low metastasis potential, respectively, were compared using AACT/SILAC coupled with SDS-PAGE-LC-MALDI -MS and a nonredundant dataset of 360 proteins were quantified as metastasis-related proteins. Expression differences were further validated by Western blotting in a series of HCC cell types with progressively increasing trend of metastasis and in clinical samples. Function annotations were also analyzed by GeneOntology, KEGG and protein-protein interactions, illustrating their biological roles in cell proliferation, apoptosis, cell migration and tumor angiogenesis.
Overall, proteome alteration in HCC and metastasis in HCC were systematically screened using AACT/SILAC, and a nonredundant protein dataset involved in HCC was identified here. The dataset contained a set of proteins related with HCC development, facilitating our understanding on the molecular mechanism of HCC and metastasis, and would benefit the early diagnosis and clinical treatment in HCC patients.
本论文以氨基酸同位素标记技术(AACT/SILAC)为技术平台,首先发展了一种判断标记肽假阳性的方法,剔除假阳性标记肽对定量的影响,改进了氨基酸同位素标记技术。最后选择了具有相同遗传学背景的两对细胞系,研究了肝癌及转移过程中蛋白质组的差异,获得一批潜在的肝癌分子标志物。此外本论文初步研究了不同搜索软件对定量结果的影响,为定量蛋白质组研究选择搜索软件提供依据(见附录)。
本论文的第一部分综述了定量蛋白质组研究的发展情况,首先对质谱仪器做了介绍,然后介绍了各种定量方法,比较了各个方法的优缺点。
本论文的第二部分改进了氨基酸同位素标记技术,并研究了同肝癌及转移有关的差异蛋白质组,具体包括:
(一)标记肽假阳性的判断
本部分发展了一种判断氨基酸同位素标记方法中标记肽假阳性的方法,计算出不同标记效率下的理论同位素分布,利用最小二乘法拟合得到标记肽的标记效率,根据假阳性阈值来判断标记肽的假阳性。本方法经实际应用,证明简单易行可靠。
(二)高转移肝癌细胞系的表达谱
蛋白质表达谱是了解肝癌的分子基础,在本实验中,利用SDS-PAGE分离结合LC-MALDI-MS鉴定技术,获得了高转移肝癌细胞系MHCC97H的蛋白质表达谱,系统分析了MHCC97H的蛋白质表达谱特点。本部分研究首先分析了表达谱的分子量和pI等,显示了本方法在鉴定具有极端分子量和pI的蛋白质方面具有的优势,发现蛋白质的细胞定位和其pI之间有一定的关联,利用GeneOntology在三个方面对表达谱蛋白做了GO注释,并利用InterPro数据库分析了表达谱的蛋白质家族特性。根据KEGG的数据和HPRD中的蛋白质相互作用数据,分析了表达谱蛋白所涉及的KEGG通路和蛋白质相互作用。
(三)肝癌差异谱的研究
本部分试验比较了肝癌细胞系QGY-7703和肝细胞系QSG-7701的蛋白质组差异,鉴定到一批和肝癌有关的蛋白质分子,这些蛋白质分子在细胞凋亡、增殖和细胞移动中发挥着重要的作用,借助Western blotting和免疫组化验证了定量结果的准确性和在临床样本中差异蛋白的表现。同时本部分研究利用GeneOntology、KEGG、蛋白质相互作用分析了差异蛋白的生物学功能,发现多个差异蛋白参与糖酵解和三羧酸循环等能量代谢,差异蛋白在MAPK、钙离子通路、Wnt等信号通路中也发挥作用。此外讨论了热激蛋白、过氧化物还原蛋白、膜联蛋白、角蛋白等在肝癌中的生物学功能。
(四)肝癌转移差异谱的研究
本部分试验利用AACT/SILAC技术比较了具有高转移能力的肝癌细胞系MHCC97H和低转移能力的肝癌细胞系MHCC97L的蛋白质组差异。利用Western blotting技术,在5个具有不同转移能力的肝癌细胞系中,检验了差异蛋白和肝癌转移能力的关系,同时也比较了差异蛋白在肝癌临床样本和癌旁组织的表达差异。利用生物信息工具,分析了差异蛋白的GeneOntology、KEGG通路和蛋白质相互作用,并在细胞增殖和凋亡、细胞移动及血管生成等方面讨论了差异蛋白的生物学功能,揭示他们在肝癌转移过程的作用。
本论文改进了氨基酸同位素标记技术,利用AACT/SILAC技术全面系统的分析了肝癌及转移过程中的蛋白质组变化,在蛋白质水平研究了肝癌中发生的一系列分子事件,探讨了肝癌的分子机制,研究和发掘一批与肝癌密切相关的蛋白质分子,必将有利于深入、系统的理解肝癌机制,促进肝癌生物标志物的发掘和临床诊断应用。
Hepatocellular carcinoma (HCC) is known as a one of difficultly curable diseases in the world and in China. Surgery remains the primary treatment of choice, but only approximately 6% of patients survive more than five years after resection. The major obstacle for the prolonging survival of HCC patients is metastasis after resection. Meanwhile the molecular mechanism underlying the HCC still has yet to be completely understood. Efforts to assess proteins involved in HCC are critical to elucidate potential biomarkers and therapy targets in HCC. Quantitative proteomics or comparative proteomics aims to screen the proteome differences associated with disease phenotype, and discovers the proteins functioning in the process of HCC. Systematic understanding using quantitative proteomics by comparing the HCC cell line versus liver cell line or by comparing HCC cell lines with different metastasis potential will show light on the molecular mechanisms of HCC and provide potential biomarkers for early diagnosis and therapy.
The present works developed one method to determine the false positive of labeled peptide in amino acid-coded mass tagging/stable isotope labeling with amino acids in cultured cell (AACT/SILAC). The works also comprehensively profiled the differential protein expression pattern using AACT/SILAC in HCC, and provided many useful biomarker candidates for HCC.
Firstly the work provided a brief summary about the development of quantitative proteomics, introducing the MS instrument and quantification methods such as 2DE、ICAT、iTRAQ and AACT/SILAC.
Secondly the work improved the AACT/SILAC technology and focused on the differential proteome of HCC, and was illustrated as following.
(1) Evaluation of the false positive of labeled peptide in AACT/SILAC
Stable isotope labeling became the chief technology in quantitative proteomics and false positive of peptide labeled was not well studied yet for the present. This article developed a method to judge the false positive of labeled peptide. Based on the probability model of isotope distribution, theoretical isotope distribution with different label efficiency were calculated, the label efficiency of peptide labeled was evaluated by comparing the experimental and theoretical isotope distribution using the least-square method. The peptide was determined as a false-positive one when its label efficiency was below the threshold. This judgment method would facilitate the precisely quantification of AACT/SILAC in proteomics.
(2) Proteome expression profile of HCC cell line with high metastasis potential
Proteome expression profile was the basis to understand the HCC mechanism and therapy. Proteins identified in HCC cell lines with high metastasis potential were put into biological context with various annotation sources followed by a bioinformatics analysis. The profile was annotated in genomic context and characterized the correlation between subcellular location and the pI. Proteins were further annotated with GeneOntology, grouped into proteins families in InterPro database and mapped to KEGG pathways they were involved. Advantages of AACT/SILAC over 2DE in identifying the proteins with high molecular weight and pI or membrane proteins were demonstrated. The protein-protein interaction network derived from HPRD was also examined.
(3) Differential proteome profile between a HCC cell line and liver cell line
Proteome alteration between two cell lines derived from HCC tissue and its adjacent tissue were screened using AACT/SILAC and differential proteome profile containing 224 proteins were obtained. Western blotting and immune histochemistry also validated the accuracy of the quantification results and the expression alteration of representative proteins in clinical HCC tissues. Biological functions of differentially expressed proteins were annotated by GeneOntology, KEGG and protein-protein interaction. These proteins were mapped to MAPK, calcium, Wnt signal pathway which regulated the cell proliferation and cell migration and functioned in HCC. Functions of annexin family, heat shock proteins and cytokeratin family in HCC were also disscused.
(4) Differential proteome profile between HCC cell lines with different metastasis potential
Proteome profile of two HCC cell lines with high and low metastasis potential, respectively, were compared using AACT/SILAC coupled with SDS-PAGE-LC-MALDI -MS and a nonredundant dataset of 360 proteins were quantified as metastasis-related proteins. Expression differences were further validated by Western blotting in a series of HCC cell types with progressively increasing trend of metastasis and in clinical samples. Function annotations were also analyzed by GeneOntology, KEGG and protein-protein interactions, illustrating their biological roles in cell proliferation, apoptosis, cell migration and tumor angiogenesis.
Overall, proteome alteration in HCC and metastasis in HCC were systematically screened using AACT/SILAC, and a nonredundant protein dataset involved in HCC was identified here. The dataset contained a set of proteins related with HCC development, facilitating our understanding on the molecular mechanism of HCC and metastasis, and would benefit the early diagnosis and clinical treatment in HCC patients.
引文
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