一种磷酸化蛋白质组学分析新方法研究

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. However, 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. We proposed here an investigation of a novel method for protein phosphorylation analysis based on the combination of a new labeling regeant to phosphor group and the related mass spectrometry. A new reagent (MPAED, 2-(4-MethylPiperazin-1 -Aacetamido) Ethanaminium Derivative) 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 MPAED will disrupt the carboxyamide bonds and form a report ion in the "quiet region" of the MS/MS spectrum. Thus we can differentiate the phosphopeptides and non-phosphopeptides by the report ion. Based on the method, we can identify all the phosphopeptides and non-phosphopeptides in one LC/MS-MS run. In this project we plan to systematically investigat and optimize this method and to apply this method in the large-scale analysis of phosphoproteins in a cell or tissue. 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.

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,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。尽管近年在磷酸化肽段的富集、磷酸化位点的鉴定和相关生物信息学方面有明显进展，但仍需要更加特异有效的分离方法和更加高灵敏度、更高通量的鉴定方法，从而实现从少量生物样品中解析全部磷酸化蛋白质的目标。本申请项目在前期探索基础上，提出基于一种全新的化学标记试剂4-甲基哌嗪乙酰乙二胺衍生物（MPAED）结合生物质谱的磷酸化蛋白质分析方法，且前期研究结果已展示了其可行性和优势。本项目将系统地开展相关方法学研究，建立对应的磷酸化蛋白质数据分析平台，并应用于重要生物学问题相关的细胞样品的大规模磷酸化蛋白质组学分析。

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，其参与生命活动几乎所有的的关键过程。细胞内蛋白磷酸化发生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。然而，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。根据这个科学问题，本课题进行了多方面的工作，建立了灵敏度高、特异性强和普适性的磷酸化蛋白质组学新方法，具体成果如下：1）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。2）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，为磷酸化定位分析奠定基础。3）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个磷酸化位点为全新的位点。本项目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。