【作者】 李茂玉；

【导师】 肖志强；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 蛋白质组学已经成为当前生命科学研究的重要内容,已广泛应用于生命科学各个领域。由于基因在转录、翻译后产生蛋白质的过程中,存在着转录水平、翻译水平的调控,同时还存在着蛋白质的翻译后修饰,因而蛋白质组研究要比基因组研究复杂得多,蛋白质组学对技术的依赖性和要求也远超过基因组学。因此,发展高通量、高灵敏度、高准确性的方法和技术一直是蛋白质组学研究面临的挑战。随着蛋白质研究技术和方法的进步,蛋白质组学研究呈现出新的特点：即由原来以蛋白质表达谱研究为中心逐步向蛋白质精确定量和功能研究转变。目前以稳定同位素标记技术为代表的定量蛋白质组学技术己逐渐成为了蛋白质组研究的新技术之一。但定量蛋白质组学技术还处于发展阶段,其研究技术和相关分析工具有待改进和完善。¹⁸O标记技术是较早应用于定量蛋白质组学研究的技术之一。该技术以酶解反应动力学原理为基础,通过酶催化反应进行蛋白质的¹⁸O标记。¹⁸O标记技术具有操作简单、条件温和、没有副产物等独特优势。但¹⁸O技术也存在如下不足：（1）在酶存在的情况下标记容易丢失,从而影响定量分析的准确性；（2）获得的数据分析相对复杂；（3）定量分析工具不足。膜蛋白在细胞与细胞识别、信号转导、物质转运等方面发挥重要功能。膜蛋白异常表达常导致细胞信号转导、细胞粘附和运动等方面的改变,并与肿瘤侵袭和转移密切相关。因此,膜蛋白质组研究已成为肿瘤蛋白质组研究的重要内容。基于上述考虑,我们首先对¹⁸O标记方法进行了优化和改进。¹⁸O标记中标记丢失是该方法的主要的弱点。针对这一点,我们采用固相胰酶作为标记酶,标记反应结束后,通过Ziptip对固相胰酶进行过滤去除,分析过滤后的样本在不同pH条件下标记的稳定性。结果显示,与热处理法比较,Ziptip过滤法能显著提高¹⁸O标记的稳定性。在连续三天的检测中未发现明显的标记丢失现象。进一步分析了Ziptip过滤法对定量分析结果的的影响,结果显示在连续三天的检测中肽段的定量差异均小于5%。对过滤样品进行等电聚焦（IEF）分离后也显示¹⁸O标记无明显丢失,进一步的分析也显示肽段标记效率与肽段分子量和理论等电点无明显相关性。另外,Ziptip过滤法所处理样品均为10-20μl,因此,我们优化和改进的¹⁸O标记方法能够用于微量蛋白样本的定量分析,这对临床稀缺样本,如激光捕获显微切割（LCM）纯化的组织样本的分析具有重要意义。我们针对目前¹⁸0蛋白定量分析工具不足的现状开发了OxyQuantToolkit工具。该工具采用java语言开发,具有跨平台的优势。分析工具的功能涵盖了¹⁸0标记技术中数据处理的多个环节,通过该工具可自动完成¹⁸0标记定量分析中的大量计算任务,分析工具可输出定量分析结果。同时分析工具还具有数据整理,转存方面的功能,这对有效管理数据也有积极作用。另外,该工具还包含了质谱数据的可视化编程包,可进行质谱通用格式文件mzxml的可视化查看。OxyQuantToolkit分析工具的开发为基于¹⁸0标记定量蛋白质组学技术的推广与应用奠定了基础。在标记方法改进和分析工具开发的基础上,我们利用改进的¹⁸0标记技术对高转移鼻咽癌细胞5-8F与不转移鼻咽癌细胞6-10B的膜蛋白组进行了定量分析。结果共鉴定了503种蛋白质,其中44%为膜蛋白,89.7%的蛋白质定量结果在0.5-2之间（均值为1.13）。对重复鉴定的395个蛋白的定量分析结果进行统计学分析,结果显示其标准差均值为0.06,具有较高的重复性。结果表明,改进的18O标记技术具有重要的应用价值。另外,参照文献标准（差异变化两倍以上、在三次重复实验中均被鉴定的蛋白质）,我们对5-8F与6-10B细胞的膜蛋白组进行了比较,获得了31个差异表达的膜蛋白,其中在5-8F中表达下调的6个,表达上调的25个。生物信息分析显示,其中一些差异蛋白如ITGB1、EphA2与细胞粘附、肿瘤侵袭和转移密切相关。这些差异蛋白质的发现为研究鼻咽癌转移机制以及鼻咽癌转移的防治提供了靶标。进一步比较5-8F与6-10B细胞的膜磷酸化蛋白组的差异,发现EphA2促转移的关键位点$897磷酸化水平在两株细胞存在差异,且EGFR能够磷酸化该位点,这提示EGFR可能通过活化EphA2促进鼻咽癌转移。本研究建立了基于Ziptip过滤去除固相胰酶的改良18O标记方法。该方法具有标记稳定、定量分析准确结果以及适合微量样本分析的特点；开发了与平台无关的基于¹⁸0标记技术的定量蛋白质组分析工具OxyQuantToolkit,为¹⁸0标记技术的推广和应用打下了基础,并开发了基于java的质谱数据可视化编程包,实现了mzxml文件的可视化浏览；采用改良的¹⁸0标记定量蛋白质组学技术比较不同转移潜能NPC细胞株的膜蛋白质组的差异,鉴定了31个与NPC转移相关的膜蛋白质,为NPC转移机制研究及其防治提供了靶标；应用定量磷酸化蛋白技术鉴定到EphA2促肿瘤关键位点S897的差异表达,证实EGFR活化能诱导EphA2促肿瘤转移的关键位点S897磷酸化,为揭示EGFR促鼻咽癌转移机制的研究提供了重要线索。更多还原

【Abstract】 Proteomics has become an important part of life science research and has been widely used in life science fields. Because there are transcriptional and translational regμlation of gene expression and post-translational modification of proteins, proteomics is much more complicated than genomics. Therefore, proteomics research is much more dependent on technology than genomic research. Therefore, the development of methods and techniques with high throughput, high sensitivity and high accuracy has been the challenge for proteomics research.With advances in the proteomics techniques and methods, proteomics research has new feature:a research focus has moved from the protein expression profile to precise quantitative and functional studies of proteins. Currently quantitative proteomics technology based on stable isotope labeling have become a new proteomics technology. However, quantitative proteomics technology is still in development stage and the research techniques need to be improved and analysis tools need to be developed.¹⁸O labeling technique is one of the earlier techniques for quantitative proteomics. The technique based on the principle of enzyme kinetics, where proteins are labeled with ¹⁸O by enzyme catalytic reaction.¹⁸O labeling technique is simple, mild technique with no by-products. However, there are some disadvantages of ¹⁸O labeling technique:（1） ¹⁸O-label is easily lost at the presence of the enzyme, thus affecting the accuracy of quantitation; （2） Data analysis is relatively complex; （3） Lack of computational tools for quantitative analysis.Membrane proteins play important roles in cell recognition, signal transduction material transport and etc. Abnormal expression of membrane proteins often lead to the changes of signal transduction, cell adhesion, motility and etc. which are related with tumor invasion and metastasis. Therefore, the study on tumors’ membrane proteome has become an important part of proteomic research.Based on the above considerations, we first optimized and improved ¹⁸O labeling method. The loss of ¹⁸O-label is the major weakness of this method. For this, We use solid-phase trypsin as a labeling enzyme and, after the reaction, the samples were filtered using Ziptip to remove solid-phase trypsin. The stability of ¹⁸O-label of the filtered samples stored under different pH conditions was analyzed. The resμlts showed that Ziptip filtration can significantly improve the stability of ¹⁸O-label, compared with the heat treatment method. There was no obvious loss of 180-label during continuous monitoring of three days. Further analysis of the impact of the Ziptip filtration on the results of quantitative analysis showed that the differences of quantitation are less than 5% during continuous monitoring of three days. There were no significant loss of ¹⁸O-label during the filter samples were separated by isoelectric focusing （IEF）. Further analysis also showed that the ¹⁸O labeled efficiency of peptides was no significant correlation with their molecμlar weight and theoretical isoelectric point. In addition, the volume of the samples handled by Ziptip was merely 10-20μl in the present study. Therefore, the optimized and improved ¹⁸O labeling method provided by us can be used for quantitative analysis of trace samples, which is of great significance for scarce clinical samples, such as laser capture microdissection （LCM） purified tissue samples and other limited samples.Considering the lack of computational tools for ¹⁸O labeling data anlaysis, We developed an computer tool named OxyQuantToolkit. The tool is developed using java language and therefore has cross-platform property. The tool has functions covering mμltiple aspects of ¹⁸O labeling data processing. A large number of computing tasks can be done automatically by the tool during ¹⁸O labeling data anslysis. The results of quantitative analysis can be output automatically. At the same time,the tool has functions for data arrangement and data dump, which has a positive effect on effective data management. In addition, the tool contains a data visualization programming package, which can be used for viewing mass spectrometry data in mzxml format. The development of OxyQuantToolkit provided the foundation for facilitating and broadening the application of ¹⁸O labeling quantitative proteomics technology.On the basis of the improvement of ¹⁸O labeling method and the development of analysis tool for ¹⁸O labeling data analysis, We use the improved ¹⁸O labeling method for the quantitative analysis of the membrane proteins from high metastatic nasopharyngeal carcinoma（NPC） 5-8F cells and non metastatic NPC 6-10B. A total of 503 proteins were identified, with 44% of membrane protein and 89.7% of the proteins’ expression ratio in 5-8F versus 6-10B were between 0.5-2 （mean 1.13）. Statistic analysis of the quantitative results of 395 proteins identified in three repeated experiments demonstrated that the average standard deviation was 0.06, showing high repeatability. The results showed that the improved ¹⁸O labeling has great applicable value. In addition, according to the standard provided in literatures （changing more than twice and identified in three repeated experiments）,31 differential expressed membrane proteins between 5-8F and 6-10B were found, of which 6 were downregμlated and 25 were upregμlated in 5-8F. Bioinformatics analysis showed that some differential expressed proteins, such as ITGB1 and EphA2, are related to cell adhesion, tumor invasion and metastasis. The discovery of these proteins provided the targets for the study of metastatic mechanisms and prevention and treatment of nasopharyngeal carcinoma. Further comparative analysis of the phosphoproteins in the membrane fraction of 5-8F and 6-10b cells found that EphA2 S897 phosphorylation site which is key to promoting metastasis are differentially expressed, and activated EGFR can phosphorylate the site, indicating that EGFR promotes NPC metastasis possibly by phosphorylating the site.An improved ¹⁸O labeling method based on removal of solid-phase trypsin using Ziptip has been established. The method has features such as high stability of label, high accuracy of quantitation and being suited to the analysis of trace samples; the platform-independent analysis tool, OxyQuantToolkit, for ¹⁸O labeling-based quantitative proteomics was developed, which provide the foundation for facilitating and broadening the application of 180 labeling technique. A java-based programming package for mass spectrometry data visualization was developed and a viewer for viewing mass spcetrometry data in mzxml format was also implemented; Comparative proteomic analysis of membrane proteins between NPC cell lines with different metastatic potential are performed using improved 180 labeling method, and 31 metastasis-related membrane proteins were identified, which provided the targets for the study of metastatic mechanism and the prevention of NPC. Comparative phsosphoproteomic analysis of membrane proteins between NPC cell lines with different metastatic potential are performed and the differential phsosphorylation level of EphA2 S897 site was identified, which is key to promoting metastasis. Further analysis confirms that activation of the EGFR can phosphorylate the site, which provide a important clue for studying the mechanism of EGFR that promoting NPC metastasis.更多还原