Sequencing Grade Tandem Mass Spectrometry for Top-Down Proteomics Using Hybrid Electron Capture Dissociation Methods in a Benchtop Orbitrap Mass Spectrometer
Sequencing Grade Tandem Mass Spectrometry for Top-Down Proteomics Using Hybrid Electron Capture Dissociation Methods in a Benchtop Orbitrap Mass Spectrometer
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DOI：
10.1021/acs.analchem.8b01901
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发表时间：
2018-09-18
影响因子：
7.4
通讯作者：
Voinov, Valery G.
中科院分区：
文献类型：
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作者：
Shaw, Jared B.;Malhan, Neha;Voinov, Valery G.
与传统的碰撞诱导解离方法相比，电子捕获解离（ECD）提供了更全面的大肽和蛋白质的表征，并且保留了不稳定的翻译后修饰。然而，ECD实验通常限于FTICR-MS的高磁场，其使得大聚阳离子和电子能够反应。在这里，我们证明了使用的电磁静ECD细胞进行ECD和混合ECD方法，利用193 nm的光子（ECuvPD）或碰撞激活（EChcD）在台式四极轨道阱质谱仪。设计了一种静电场电子捕获器（ECD），以取代四极和C阱之间的转移八极。该实施方式使得能够容易地安装ECD池，并且离子可以独立地经受ECD、UVPD、HCD或任何组合。使用泛素（8.6 kDa）进行ECD、ECuvPD和EChcD片段化倾向的初始基准和表征。ECD产生了广泛的序列覆盖率低电荷状态的泛素以及较大的蛋白质碳酸酐酶II（29 kDa），表明假活化离子条件。此外，相对高数量的d-和w-离子能够区分同量异位异亮氨酸和亮氨酸残基，并表明电子能量的分布产生热ECD型片段化。我们报告了迄今为止最全面的表征高达29 kDa的模型蛋白和单克隆抗体在亚基水平。对于碳酸酐酶II（29 kDa），ECD、ECuvPD和EChcD分别产生93、95和91%的序列覆盖率，单克隆抗体亚基的靶向在线分析产生86%的总体抗体序列覆盖率。
Compared to traditional collision induced dissociation methods, electron capture dissociation (ECD) provides more comprehensive characterization of large peptides and proteins as well as preserves labile post translational modifications. However, ECD experiments are generally restricted to the high magnetic fields of FTICR-MS that enable the reaction of large polycations and electrons. Here, we demonstrate the use of an electromagnetostatic ECD cell to perform ECD and hybrid ECD methods utilizing 193 nm photons (ECuvPD) or collisional activation (EChcD) in a benchtop quadrupole-Orbitrap mass spectrometer. The electromagnetostatic ECD cell was designed to replace the transfer octapole between the quadrupole and C-trap. This implementation enabled facile installation of the ECD cell, and ions could be independently subjected to ECD, UVPD, HCD, or any combination. Initial benchmarking and characterization of fragmentation propensities for ECD, ECuvPD, and EChcD were performed using ubiquitin (8.6 kDa). ECD yielded extensive sequence coverage for low charge states of ubiquitin as well as for the larger protein carbonic anhydrase II (29 kDa), indicating pseudo-activated ion conditions. Additionally, relatively high numbers of d- and w-ions enable differentiation of isobaric isoleucine and leucine residues and suggest a distribution of electron energies yield hot-ECD type fragmentation. We report the most comprehensive characterization to date for model proteins up to 29 kDa and a monoclonal antibody at the subunit level. ECD, ECuvPD, and EChcD yielded 93, 95, and 91% sequence coverage, respectively, for carbonic anhydrase II (29 kDa), and targeted online analyses of monoclonal antibody subunits yielded 86% overall antibody sequence coverage.