在线酶解-全二维压力辅助毛细管电色谱系统的构建及高通量蛋白质组分析研究

Large-scale, high throughput separation and subsequent identification of complex proteins still remain great challenge in current proteomics. Although two-dimensional gel electrophoresis is widely used method for the analysis of complex protein mixtures, it often suffers from some drawbacks such as sample loss, time-consuming and difficulty in automation and so on. Recently, different types of multidimensional separation systems based on liquid chromatography and capillary electrophoresis have been developed and regarded as a powerful separation technique for its large peak capacity, effective separation and high resolution, However, it is rather challenging to make a balance between resolution, analysis throughput and peak capacity. To address these problems, a highly integrated platform of comprehensive two-dimensional pressure-assisted capillary electrochromatography coupled to online immobilized enzyme reactor is proposed for high throughput and efficient proteome analysis in this proposal. The missions of this proposal included as follows: (1) based on the principle of interface/nanoconfined catalytic system, developing a series of enzyme-inorganic/enzyme-metal organic framework hybrid monolithic capillary-based immobilized enzyme reactors for rapid, high throughput and online digestion of proteins; (2) developing a series of organic-inorganic hybrid monolithic columns with mixed mode for high efficient separation of proteins and peptides; (3) based on the above achievement, developing an integrated platform containing two-dimensional protein/peptide separation, online protein digestion and UV/Vis or MS identification units. Through the implementation of the project, it can be expected to achieve more comprehensive identification of proteins, improving the resolution, throughput and peak capacity. More importantly, it can also be expected to provide a strong technical support and scientific instrument for proteomics in future.

规模化、高通量的蛋白质分离和鉴定依然是当前蛋白质组学研究的重点和难点。尽管现有的多维色谱技术解决了传统双向电泳在蛋白质分析中存在的样品易丢失、分析时间长以及难以自动化等问题，但在色谱分辨率、分析通量以及峰容量等方面依旧难以兼顾。针对上述问题，本项目拟开展在线酶解-全二维压力辅助毛细管电色谱系统构建及蛋白质组高效、高通量分离分析研究，其核心技术包括:（1）基于界面/纳米限域酶催化原理，研制酶-无机盐/酶-金属有机骨架杂化毛细管微酶反应器，发展高通量、快速的在线酶解技术；（2）结合“一锅同步点击化学”技术，制备有机-无机杂化毛细管电色谱整体柱，发展高效的蛋白质/肽段分离分析方法；（3）集成蛋白质分离、在线酶解、肽段分离等单元，构建在线酶解-全二维压力辅助毛细管电色谱系统。通过本项目的研究，有望实现高分辨率、高通量、高峰容量的蛋白质组分离分析，为后续的蛋白质组学研究提供坚实的仪器和技术支撑。

以经典双向凝胶电泳（2-DE）为基础的蛋白质组分析技术存在明显的局限性，严重制约着蛋白质组学研究的发展进程。针对上述问题，项目围绕在线酶解-全二维压力辅助毛细管电色谱系统的构建，重点开展了酶反应器制备、色谱整体柱及色谱固定相制备、以及基于生物质谱的蛋白质高通量鉴定等方面开展系统研究。经过4年的实施，项目在以下几个方面取得进展：（1）利用界面或纳米限域酶催化原理，开发了酶-无机盐杂化和酶-金属有机骨架杂化的固定化酶反应器，建立了基于这两类酶反应器的蛋白质快速在线/离线酶解技术，实现了组学样品中蛋白质的高通量、快速酶解；（2）结合“一锅同步点击化学”技术，制备了一系列新型的有机-无机杂化毛细管整体柱，发展了高效的蛋白质/肽段分离新方法; 结合纳米技术，制备核-壳结构硅球共价有机框架以及球形共价有机框架色谱固定相，实现了蛋白质/多肽等生物分子的高效、快速分离；（3）制备了一系列磁性纳米复合材料，实现了低丰度翻译后修饰蛋白质/内源性肽等生物标志物的高效、高选择性富集；（4）开发了一系列基质辅助/表面增强激光解吸离子化质谱技术，实现了低分子肽段的高通量筛查与高灵敏检测；（5）在此基础上，整合蛋白质预富集、分离、在线酶解以及质谱鉴定技术，分别构建了二维微柱色谱分离分析平台以及常压电离质谱检测平台，初步实现了复杂生物体系中蛋白质的高效分离与快速酶解，为后续蛋白质组的高通量鉴定提供了技术支撑。该项目部分研究成果获2020年度中国分析测试协会科学技术特等奖。累计发表论文26篇，其中SCI论文24篇，中文核心2篇。具体包括J. Am. Chem. Soc., Mass Spectrom. Rev., Anal. Chem., Chem. Commun., ACS Appl. Mater. Interfaces,《科学通报》，《色谱》等，研究成果多次以封面形式进行报道。申请国家发明专利8项，其中已授权3项。

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