Zhao Yimeng, Riley Nicholas M, Sun Liangliang, Hebert Alexander S, Yan Xiaojing, Westphall Michael S, Rush Matthew J P, Zhu Guijie, Champion Matthew M, Mba Medie Felix, Champion Patricia A DiGiuseppe, Coon Joshua J, Dovichi Norman J
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
‡Department of Biomolecular Chemistry, Genome Center of Wisconsin, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
Anal Chem. 2015;87(10):5422-9. doi: 10.1021/acs.analchem.5b00883. Epub 2015 May 6.
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 the Mycobacterium marinum bacterial 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.
自上而下的蛋白质组学为完整蛋白质的表征提供了可能，但这种方法仍面临许多挑战，包括高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）在完整蛋白质的分离方面显示出巨大潜力，特别是对于同一基因产物的差异修饰蛋白变体。然而，迄今为止，CZE仅与基于碰撞的碎片化方法联用。在此，我们报道了首次将电子转移解离（ETD）与在线CZE分离联用进行自上而下的蛋白质组学研究，分析了四种标准蛋白质的混合物以及来自海分枝杆菌细菌分泌组的复杂蛋白质混合物。在一台轨道阱精英系统上使用多功能解离池，我们证明CZE与ETD以及高能碰撞解离（HCD）完全兼容，并且这两种互补的碎片化方法可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），一种最近引入的用于增强ETD碎片化的方法，在CZE分离中表现出良好性能，可大大提高蛋白质表征。当与HCD结合使用时，AI-ETD使标准混合物和复杂混合物中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为CZE分离带来的益处。
