Coupling capillary zone electrophoresis with electron transfer dissociation and activated ion electron transfer dissociation for top-down proteomics.
Coupling capillary zone electrophoresis with electron transfer dissociation and activated ion electron transfer dissociation for top-down proteomics.
复制标题
将毛细管区带电泳与电子转移解离和活化离子电子转移解离结合用于自上而下的蛋白质组学。
DOI：
10.1021/acs.analchem.5b00883
复制
发表时间：
2015
影响因子：
7.4
通讯作者：
Dovichi,NormanJ
中科院分区：
文献类型：
--
作者：
Zhao,Yimeng;Riley,NicholasM;Sun,Liangliang;Hebert,AlexanderS;Yan,Xiaojing;Westphall,MichaelS;Rush,MatthewJP;Zhu,Guijie;Champion,MatthewM;MbaMedie,Felix;Champion,PatriciaADiGiuseppe;Coon,JoshuaJ;Dovichi,NormanJ
自上而下的蛋白质组学提供了完整蛋白质表征的潜力，但这种方法仍然存在许多挑战，包括有效的蛋白质分离和完整蛋白质的有效碎片化。毛细管区带电泳（CZE）在分离完整蛋白，特别是同一基因产物的差异修饰蛋白形态方面显示出巨大的潜力。然而，到目前为止，CZE仅用于基于碰撞的碎片化方法。在这里，我们报告了电子转移解离（ETD）与在线CZE分离自上而下的蛋白质组学的首次实施，分析了来自海洋分枝杆菌分泌组的四种标准蛋白和一种复杂蛋白混合物。利用Orbitrap Elite系统上的多用途解离细胞，我们证明了CZE与ETD以及高能碰撞解离（HCD）完全兼容，并且这两种互补的分裂方法可以在电泳时间尺度上串联使用，以改善蛋白质表征。此外，我们表明，活化离子电子转移解离（AI-ETD）是最近引入的一种增强ETD碎片化的方法，它与CZE分离提供了有用的性能，大大提高了蛋白质的表征。当与HCD结合使用时，AI-ETD将标准和复杂混合物中蛋白质的蛋白质序列覆盖率提高了200%以上，突出了电子驱动解离方法可以增加CZE分离的好处。
Top-down proteomics offers the potential for full protein characterization, but many challenges remain for this approach, including efficient protein separations and effective fragmentation of intact proteins. Capillary zone electrophoresis (CZE) has shown great potential for separation of intact proteins, especially for differentially modified proteoforms of the same gene product. To date, however, CZE has been used only with collision-based fragmentation methods. Here we report the first implementation of electron transfer dissociation (ETD) with online CZE separations for top-down proteomics, analyzing a mixture of four standard proteins and a complex protein mixture from theMycobacterium marinumbacterial secretome. Using a multipurpose dissociation cell on an Orbitrap Elite system, we demonstrate that CZE is fully compatible with ETD as well as higher energy collisional dissociation (HCD), and that the two complementary fragmentation methods can be used in tandem on the electrophoretic time scale for improved protein characterization. Furthermore, we show that activated ion electron transfer dissociation (AI-ETD), a recently introduced method for enhanced ETD fragmentation, provides useful performance with CZE separations to greatly increase protein characterization. When combined with HCD, AI-ETD improved the protein sequence coverage by more than 200% for proteins from both standard and complex mixtures, highlighting the benefits electron-driven dissociation methods can add to CZE separations.