【作者】 熊继先；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 蛋白质磷酸化修饰是最常见,最重要的的一种蛋白质翻译后修饰方式,蛋白质磷酸化和去磷酸化几乎调节着生命活动的所有关键过程。同时磷酸化的失调会导致很多严重的人类疾病,如癌症,糖尿病,心脏病,老年痴呆症等。因此,蛋白质磷酸化研究一直以来都是蛋白质组学研究的热点,同时用蛋白质组学的策略研究生物体的磷酸化修饰也是功能蛋白质组学的重要研究内容。由于磷酸化蛋白质在生物体内的含量很低,对它的检测和位点分析一直是蛋白质组学的难点,而磷酸化蛋白质的大规模化分析和位点鉴定更是磷酸化蛋白质组研究面临的巨大挑战。神经干细胞在生命活动中执行了多种重要的生理功能,具有重要的理论意义和广阔的应用前景,因此其磷酸化研究有助于人们深入的理解和认识神经干细胞的复杂功能以及蛋白质磷酸化在神经干细胞增殖,生长等生物过程中的意义。本论文的工作包括3个方面：1)基于一种全新的化学标记试剂和生物质谱的磷酸化蛋白质分析方法的建立；2)磷酸化蛋白质数据分析平台的建立；3)大规模磷酸化蛋白质组学分析技术在小鼠神经干细胞磷酸化蛋白质组学研究中的应用。第一部分以标准磷酸化蛋白质为样本建立了蛋白质磷酸化修饰分析技术体系：自主设计并合成了一种新的磷酸化特异性标记试剂MPAE(4—甲基哌嗪乙酰乙二胺)；通过对肽段的酯化,标记有效的将标记试剂引入到磷酸化基团；最后用串联质谱分析区分磷酸化肽段和非磷酸化肽段,并结合数据库检索鉴定确定磷酸化位点。第二部分建立了磷酸化蛋白质组学数据分析及其处理平台,通过对搜库参数进行了优化,确立了严格的搜寻条件,保证了结果的可靠性和准确性,为大规模,高通量的磷酸化蛋白质组学分析奠定了基础,此外,我们编写了适合我们实验方法的磷酸化定位程序,为接下来的磷酸化定位分析铺平了道路。第三部分利用所建立的磷酸化蛋白质分析方法大规模的分析了小鼠神经干细胞(C17.2 cell)提取的全蛋白质中的磷酸化蛋白质,共鉴定出337个磷酸化肽,405个磷酸化位点,分别归属于259个磷酸化蛋白质,其中有18个磷酸化位点是文献中没有报道过的。以上信息的获得为探索干细胞增殖及其特性的保持机制,寻找一批与神经肿瘤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。总之,本研究系统的提出了一套新的磷酸化蛋白质组学分析方法,为磷酸化蛋白质组学研究提供了新的思路。更多还原

【Abstract】 Protein phosphorylation is one of the most important post-translational modifications in cells, and the reversible phosphorylation of proteins regulates nearly every aspect of cell life from signaling transduction, metabolism, gene’s expression to cell growth, division, differentiation and development. Moreover, the disregulation of protein phosphorylation will induce many human diseases, most notable cancer, diabetes, heart disease, Alzheimer’s disease, and so on. So phosphoproteomics has been a hotspot in proteomics research and detecting phosphoproteins and defining their phosphorylation sites are very important to understand the mechanism by which phosphorylated protein affects a biological pathway. However, because the phosphoprotein content is generally low, analysis of phosphoproteins is not straightforward and a large-scale analysis of phosphoproteins in a cell or tissue is still a big technical challenge in phosphoproteomic research. Neural stem cell performs many complex and essential functions, which are most involved reversible phosphorylation regulation. The phosphoproteome analysis of the neural stem cell allows us to further understand and recognize the complex function and the essential regulation of phosphoprotein in division, growth and development of neural stem cells.Our research contain following three parts:1) Developing methods for protein phosphorylation analysis based on the combination of a new labeling regeant to phosphor group and the related mass spectrometry; 2) Constructing the data analysis platform for the large scale phosphoproteomics; 3) Analyzing the phosphoproteomics of proteins from neural stem cells.In the first part, a systematic analysis methods for phosphoproteins has been established. The standard protein digest mixtures were converted to the corresponding methyl esters (that is, CH30-) at first. Then the methylated peptides were subjected to a one-pot reaction in the presence of carbodiimide (that is, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide; EDC), imidazole and a reagent (MPAE,2-(4-MethylPiperazin-1-Aacetamido)Ethanaminium) which was synthesized in this lab and was designated to label and indicate phosphate groups in the process of MS analysis. When MS/MS analysis, the phosphopeptides which are labeled by MPAE will disrupt the carboxyamide bonds and form a report ion, which is 113 in the "quiet region" of the MS/MS spectrum. Thus we can differentiate the phosphopeptides and non-phosphopeptides by the report ion(113 in the MS/MS spectrum). Based on the method, we can identify all the phosphopeptides and non-phosphopeptides in one LC/MS-MS run.In the second part, the data analysis platform for the large scale phosphoproteomics is established. The database search parameters are optimized to make sure that the result is precise and reliable. It can lay the foundation for the large scale, high-throughput phosphoproteomics analysis. A phosphorylation site location method and procedure for our experiment has been developed, and it is convenient for the phosphorylation site analysis of proteins from neural stem cell line.In the third part, a robust and automatic system was developed, and phosphoproteomics analysis of proteins from neural stem cell line(C17.2 cell line) are reported here. A total of 259 phosphoproteins,337 phosphopeptide sequences are determined,407 phosphorylation sites are defined, and 18 of those sites are newly identified. These results would allow further insight into the mechanism of stem cell’s division and growth and may help to search some biomarkers for neural tumor.In total, a noval system strategy was developed for phosphoproteomics. These methods in our strategy can be used for analysis of phosphoproteomes of other proteome samples.更多还原