中文摘要
发展蛋白质多维分离、在线酶解以及质谱鉴定的集成化技术平台对于提高蛋白质组高通量研究的准确度和可信度至关重要。为解决该平台中存在的色谱流动相与蛋白质酶解环境不匹配以及基于在线原位聚合整体材料的酶反应器刚性不足的问题，本项目新型有机纤维素膜接口，能够将1M的盐溶液置换到10 mM以下；制备了无机-有机杂化材料介质的微酶解反应器，能够在30 s和50 s内完成标准蛋白质和复杂蛋白质混合物的酶解；将制备的新型微酶解反应器以及有机膜接口，结合构建的SEC/RPLC蛋白质多维分离系统、多肽反相分离系统以及质谱，构建了集蛋白质分离、在线除盐、在线酶解、多肽分离以及质谱鉴定的一体化蛋白质分离鉴定平台。将其应用于鼠脑提蛋白质的分离鉴定，经3次分析，共鉴定了1476种蛋白质，整个系统峰容量可达5670。发表SCI论文5篇，申请专利3项。
英文摘要
The development of multidimensional separation combined with on-line digestion and MS/MS identification is important for enhancement of the high throughput analysis of proteins. However there are still many problems in the whole platform,such as the mobile phase is unfrindly to the digestion of proteins, and also the monolith of immobilized enzyme reactor(IMER) is not strong enough to perform in the whole analysis.In this project the membrane interface was used to dialyze the salts in samples and using this interface 1M salts could be dialyzed under 10mM.And also The oranic-inorganic hybrid silica monolith was used as the matrix of IMER,as a result the standard protein and the mixture of proteins could be digested within 30s and 50s.Finally an on-line platform involving two dimensional SEC-RPLC separation of proteins,on-line dialysis, on-line digestion and RPLC-MS/MS analysis was established.This platform was used for the mouse brain proteins analysis.As a result 1476 proteins were identified,and the peak capacity was 5670..In this project we published 5 papers embodied by SCI and applied 3 patents.
结题摘要
发展蛋白质多维分离、在线酶解以及质谱鉴定的集成化技术平台对于提高蛋白质组高通量研究的准确度和可信度至关重要。为解决该平台中存在的色谱流动相与蛋白质酶解环境不匹配以及基于在线原位聚合整体材料的酶反应器刚性不足的问题，本项目新型有机纤维素膜接口，能够将1M的盐溶液置换到10 mM以下；制备了无机-有机杂化材料介质的微酶解反应器，能够在30 s和50 s内完成标准蛋白质和复杂蛋白质混合物的酶解；将制备的新型微酶解反应器以及有机膜接口，结合构建的SEC/RPLC蛋白质多维分离系统、多肽反相分离系统以及质谱，构建了集蛋白质分离、在线除盐、在线酶解、多肽分离以及质谱鉴定的一体化蛋白质分离鉴定平台。将其应用于鼠脑提蛋白质的分离鉴定，经3次分析，共鉴定了1476种蛋白质，整个系统峰容量可达5670。发表SCI论文5篇，申请专利3项。