Optimizing macromolecular tandem mass spectrometry of large non-covalent complexes using heavy collision gases
Optimizing macromolecular tandem mass spectrometry of large non-covalent complexes using heavy collision gases
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DOI：
10.1016/j.ijms.2007.06.012
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发表时间：
2007-12-01
影响因子：
1.8
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
--
作者：
Lorenzen, Kristina;Versluis, Cees;Heck, Albert J. R.
我们评估和优化条件的串联质谱的大分子蛋白质复合物，使用改进的Q-ToF仪器改变气体压力内的六极碰撞池，并研究了使用不同的碰撞气体，如氩气，氪和氙气的影响。这些变化影响了离子通过仪器的传输和解离效率。特别是使用较重的氙作为碰撞气体，更容易产生高质量碎片离子，并更有效地传输。我们使用800 kDa GroEL 14-mer分子伴侣机制进行这些初步研究。应用优化的串联质谱条件，即相对较高的压力和氙作为碰撞气体，我们详细研究了一个和两个gp 5底物蛋白的结合对伴侣蛋白复合物的气相解离的影响，构建分解图。我们观察到底物多肽的结合对伴侣蛋白复合物的气相稳定性具有显著影响，其中含有一个和两个底物分子的复合物比无底物GroEL更不易于解离。有趣的是，GroEL：gp 5和GroEL：（gp 5）（2）复合物仅通过GroEL单体离子的消除而解离，表明两种gp 5底物可能分别包封在GroEL的顺式和反式环的两个空腔内。从获得的所有数据，我们得出结论，较重的氙是非常大的大分子复合物的串联质谱的首选碰撞气体。(C)2007年由Elsevier B. V.出版。
We evaluated and optimized conditions for tandem mass spectrometry on macromolecular protein complexes, using a modified Q-ToF instrument varying the gas pressure inside the hexapole collision cell, and studied the influence of using different collision gases, e.g. argon, krypton and xenon. These variations affected ion transmission through the instrument and dissociation efficiency In particular the high mass fragment ions were generated more readily and transmitted more efficiently using the heavier xenon as collision gas. We used the 800 kDa GroEL 14-mer chaperone machinery for these initial studies. Applying the optimized tandem mass spectrometry conditions, i.e. relatively high pressures and xenon as collision gas, we studied in detail the influence the binding of one and two gp5 substrate proteins had on the gas-phase dissociation of the chaperonin complex constructing break-down diagrams. We observed that the binding of substrate polypeptides had a significant effect on the gas-phase stability of the chaperonin complex, with the complex containing one and two substrate molecules being less susceptible towards dissociation than the substrate free GroEL. Interestingly, the GroEL:gp5 and GroEL:(gp5)(2) Complexes dissociate exclusively via the elimination of GroEL monomeric ions, indicating that both gp5 substrates are likely encapsulated inside the two cavities of, respectively, the cis and trans ring of the GroEL. From all data acquired, we conclude that the heavier xenon is the preferred collision gas for tandem mass spectrometry on very large macromolecular complexes. (C) 2007 Published by Elsevier B.V.