Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.
Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.
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DOI：
10.1021/acs.analchem.9b04763
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发表时间：
2020-03-03
影响因子：
7.4
通讯作者：
Lermyte F
中科院分区：
文献类型：
--
作者：
Williams JP;Morrison LJ;Brown JM;Beckman JS;Voinov VG;Lermyte F
基于电子的片段化方法已经彻底改变了生物分子质谱，特别是天然和自上而下的蛋白质分析。在这里，我们报告使用一种新的电磁静电池进行电子捕获解离（ECD）内的四极杆/离子迁移率/飞行时间质谱仪。该单元安装在仪器的离子迁移率和飞行时间区域之间，并且碎片化足够快以与迁移率分离兼容。在修改之前，该仪器已经在四极和迁移率区域之间配备了电子转移解离（ETD）。我们表现出优异的变性肽和蛋白质的碎片效率，而不需要在气相中捕获离子。此外，我们证明了非共价蛋白质复合物的自上而下的骨架片段化，导致与使用仪器现有的ETD能力所实现的序列覆盖率相当。有限的碰撞离子激活的血红蛋白四聚体前ECD反映在所观察到的碎片模式，和互补的离子迁移率测量前ECD提供正交的证据单体展开在这个复杂的。这里展示的方法为自上而下的蛋白质组学和基于质谱的结构生物学研究提供了一个强大的平台。
Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument’s existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.
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影响因子：
7.4
作者：
Brodbelt JS
通讯作者：
Brodbelt JS
影响因子：
15
作者：
Breuker, K;Oh, HB;McLafferty, FW
通讯作者：
McLafferty, FW
影响因子：
7.4
作者：
Lacki, Mateusz K.;Lermyte, Frederik;Gambin, Anna
通讯作者：
Gambin, Anna
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作者：
Lermyte, Frederik;Verschueren, Tim;Sobott, Frank
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Sobott, Frank
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7.4
作者：
Robb, Damon B.;Brown, Jeffery M.;Blades, Michael W.
通讯作者：
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