Native electrospray and electron-capture dissociation FTICR mass spectrometry for top-down studies of protein assemblies.
Native electrospray and electron-capture dissociation FTICR mass spectrometry for top-down studies of protein assemblies.
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DOI：
10.1021/ac200695d
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发表时间：
2011-07-15
影响因子：
7.4
通讯作者：
Gross, Michael L.
中科院分区：
文献类型：
--
作者：
Zhang, Hao;Cui, Weidong;Wen, Jianzhong;Blankenship, Robert E.;Gross, Michael L.
质谱的高灵敏度、扩展的质量范围和快速数据采集/处理及其与天然电喷雾电离（ESI）的耦合使得该组合与蛋白质分析的其他生物物理方法互补。分子量高达MDa的蛋白质组装体现在可以通过这种方法获得。目前大多数方法都使用四极杆/飞行时间串联质谱，有时与离子迁移率相结合，以揭示化学计量，形状和蛋白质组装体的解离。然而，亚基的氨基酸序列仍然严重依赖于独立的自下而上的蛋白质组学。我们在这里描述了一种方法来研究蛋白质组装，集成了电子捕获解离（ECD），天然ESI和FTICR质谱（12特斯拉）。酵母醇脱氢酶（147 kDa）、伴刀豆球蛋白A（103 kDa）和光合Fenna-Matthews-Olson天线蛋白复合物（140 kDa）的组装亚基的柔性区域可以通过ECD或“活化离子”ECD测序。此外，还可以确定ConA组装体的非共价金属结合位点。最重要的是，经历片段化的区域，无论是从末端之一通过ECD或从蛋白质的中间，如由CID引发的，与来自该蛋白质的X射线晶体学的B因子很好地相关。该因子是原子可以从其配位位置移动的程度的量度，作为温度或晶体缺陷的函数。该方法不仅提供了自上而下的复杂亚基的蛋白质组学信息，而且还提供了与离子迁移率所获得的信息互补的结构见解。
The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 Tesla). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna-Matthews-Olson antenna protein complex (140 kDa) can be sequenced by ECD or “activated-ion” ECD. Furthermore, non-covalent metal-binding sites can also be determined for the ConA assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.
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通讯作者：
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