面向二硫键应用的三肽交联质谱技术研究

Chemical crosslinking of proteins coupled with mass spectrometry analysis (CXMS) takes advantage of mass spectrometry to get the information of protein structures and protein-protein interactions. Comparing with the traditional technologies, CXMS has lower protein purity requirement, lower cost and much higher throughput. Our team has spent about three years on the research of CXMS technology, and has developed the pLink software system, which has been published on Nature Methods in 2012. However, a more complex form, triple-peptide crosslinking, which is of great significance to disulfide bond identification, is rarely studied yet. Therefore, further research is needed to make a deeper investigation into the triple-peptide crosslinking technology, which will stimulate the application of CXMS in more biological problems. This project will mainly deal with the following three problems: (1) Algorithm. Facilitate the triple-peptide crosslinking identification by fragmentation pattern discovery, scoring function design, accelerating research and evaluation methods study; (2) Software. Develop a software system for the triple-peptide crosslinking data analysis; (3) Application. Optimize the system for disulfide bond identification. We expect this work to be the first one that provides a complete solution to disulfide bond identification problem, and pLink will be downloaded more than 300 times.

交联质谱技术将质谱技术与交联技术相结合，在研究蛋白质结构与相互作用上有着速度快、成本少、蛋白质纯度要求低等优势。申请人团队经过三年的合作探索，研发了自主知识产权的二肽交联鉴定软件系统pLink，并于2012年发表于Nature Methods期刊。然而，二硫键鉴定中广泛存在、具有重要研究意义的三肽交联鉴定，在国际国内普遍未得到重视。为完善三肽交联质谱技术，扩展二硫键研究应用，本项目将主要围绕以下三个关键问题展开：(1) 算法研究，从碎裂规律发现、打分设计、加速算法研究和评价设计四个方面深入探索三肽交联鉴定问题；(2) 软件系统开发，实现易用有效的三肽交联鉴定系统；(3) 生物学应用研究，针对二硫键交联鉴定进行探索与改进。我们期望在国际上率先比较完整地解决二硫键鉴定问题，pLink软件国内外注册下载达到300套。

本项目研究了蛋白质组二硫键二肽/三肽交联质谱鉴定问题，先后完成pLink-SS、pLink 2和pLink 3三个软件。pLink-SS在化学交联质谱鉴定引擎pLink的基础上，通过构建合成肽段二硫键二肽交联质谱标注数据集，分析了二硫键二肽交联质谱碎裂规律，首次实现了蛋白质组水平上的二硫键鉴定，研究文章于2015年发表在Nature Methods，国内外同行利用pLink-SS软件所做研究发表文章10篇（其中含Nature一篇、Nature Comms一篇）。pLink 2在pLink-SS基础上引入肽段碎片离子索引技术实现40倍加速，引入模拟数据集搜索、标注数据集加陷阱库搜索和15N标记检验实现全面的精度评测，其鉴定流程文章于2018年发表在Biophysics Reports，研究文章于2018年底投稿Nature Communications审毕修改中，软件于2018年元旦发布、全年注册下载759（其中国外490）。pLink 3在pLink-SS、pLink 2基础上，通过构建合成肽段二硫键三肽交联质谱标注数据集，分析了二硫键三肽交联质谱碎裂规律，在碎片离子索引之外进一步引入序列片段索引以提升速度，导出了三肽交联TDA-FDR公式以控制错误率，首次实现了蛋白质组水平上的二硫键三肽交联质谱鉴定。pLink-SS、pLink 2、pLink 3系列软件代表蛋白质组学二硫键质谱鉴定当前最高水平。