蛋白质组末端肽的深度覆盖定性和高准确度定量新技术研究

项目名称： 蛋白质组末端肽的深度覆盖定性和高准确度定量新技术研究

项目编号： No.21475127

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 数理科学和化学

项目作者： 单亦初

作者单位： 中国科学院大连化学物理研究所

项目金额： 86万元

中文摘要： 针对具有高度序列特异性和重要生物功能的蛋白质末端肽，在组学水平上发展富集、分离、鉴定和定量分析的新技术，有望显著降低蛋白质组样品的复杂度,实现对具有重要生物意义低丰度蛋白质的可信鉴定、准确定量和生物学验证。因此，本项目拟开展蛋白质组末端肽的深度覆盖定性和高准确度定量新技术研究。通过发展适合于多种酶切方式的高效和高选择性蛋白质组C末端和N末端肽富集方法，利用多酶酶解产生的不同末端肽以及C末端和N末端肽的互补性，提高鉴定覆盖率；通过对末端肽进行连续离子监测，以具有高特异性的二级质谱碎片离子强度为丰度特征，提高蛋白质组末端肽定量的准确度；以蛋白质末端肽为抗原决定基，合成高特异性的抗体，对目标蛋白质进行亲和富集和生物学验证。此外，还将上述新技术应用于人肝癌高低转移细胞株蛋白质组的差异分析和生物学验证，以期为揭示肿瘤转移机理和实现早期诊断提供重要技术支撑。

中文关键词： 末端肽；定量；多酶酶切；连续离子监测；肝癌转移

英文摘要： Development of novel techniques for the enrichment, separation, identification and quantification of protein terminal peptides with high specific sequence and important biological functions is of great promise to significantly reduce the complexity of proteome samples and realize the confident identification, accurate quantification and biological validation of low abundant proteins of biological importance. Hence, the aim of this project is to develop novel techniques for the deep-coverage identification and high accurate quantification of proteome terminal peptides. Firstly, develop novel method with high efficiency and selectivity which is capable of enriching N and C terminal peptide of proteome sample digested by various kinds of enzyme, improve the identification coverage of protein terminal peptides taking use of the complementarity of N and C terminal peptides generated by different enzyme; Secondly, develop continuous ion monitoring method for the identification of terminal peptides and utilize MS/MS fragment ions intensity as the abundance feature to improve the quantification accuracy for proteome terminal peptides; Thirdly, carry out affinity purification and biological validation for target proteins by using the protein terminal peptide as epitope to synthesize antibody with high specificity. Lastly, apply the above techniques to the proteome differential analysis and biological validation of human liver cancer cell lines with high and low metastatis rate, which will provide important support for the mechanism exploration and early diagnosis of cancer metastatis.

英文关键词： terminal peptide;quantification;multi-enzyme digestion;continuous ion monitoring;cancer metastatis