Extended Gas-Phase Trapping Followed by Surface-Induced Dissociation of Noncovalent Protein Complexes
Extended Gas-Phase Trapping Followed by Surface-Induced Dissociation of Noncovalent Protein Complexes
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DOI：
10.1021/acs.analchem.5b03479
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发表时间：
2016-01-19
影响因子：
7.4
通讯作者：
Wysocki, Vicki H.
中科院分区：
文献类型：
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作者：
Harvey, Sophie R.;Yan, Jing;Wysocki, Vicki H.
质谱已成为研究蛋白质和蛋白质复合物的有用工具。探索蛋白质和蛋白质复合物的结构如何受到溶剂缺乏的影响，以及如何随着气相中时间的增加而改变，这是一个根本的兴趣。在这里，我们证明了一系列的蛋白质和蛋白质复合物可以被限制在陷阱T-波区域内的一个修改后的沃茨Synapt G2 S仪器，包括单体（β-乳球蛋白），二聚体（β-乳球蛋白和烯醇化酶），四聚体（链霉亲和素，伴刀豆球蛋白A，和丙酮酸激酶），和五聚体（C-反应蛋白）复合物，大小范围高达237 kDa。我们证明，复合物可以被限制在陷阱区域内的不同长度的时间在1-60秒的范围内，并与高达86%的捕获效率为1秒的捕获。此外，使用模型系统，我们表明，这些非共价复合物也可以通过表面诱导解离（SID）的捕获后片段化。SID揭示了类似的解离模式，在所有的捕获时间研究未活化的蛋白质复合物，这表明在这个时间尺度上发生的任何构象变化是不够的，以造成实质性的差异，这些复合物的SID光谱。有意改变结构锥激活产生一个独特的SID光谱，观察到的差异被保存，相比未激活的复合物，捕获后。然而，也观察到作为捕获时间的函数的活化复合物的SID光谱的细微差异。
Mass spectrometry has emerged as a useful tool in the study of proteins and protein complexes. It is of fundamental interest to explore how the structures of proteins and protein complexes are affected by the absence of solvent and how this alters with increasing time in the gas phase. Here we demonstrate that a range of protein and protein complexes can be confined within the Trap T-wave region of a modified Waters Synapt G2S instrument, including monomeric (beta-lactoglobulin), dimeric (beta-lactoglobulin and enolase), tetrameric (streptavidin, concanavalin A, and pyruvate kinase), and pentameric (C-reactive protein) complexes, ranging in size up to 237 kDa. We demonstrate that complexes can be confined within the Trap region for varying lengths of time over the range 1-60 s and with up to 86% trapping efficiency for 1 s trapping. Furthermore, using model systems, we show that these noncovalent complexes can also be fragmented by surface-induced dissociation (SID) following trapping. SID reveals similar dissociation patterns over all trapping times studied for unactivated protein complexes, suggesting that any conformational changes occurring over this time scale are insufficient to cause substantial differences in the SID spectra of these complexes. Intentional alteration of structure by cone activation produces a distinct SID spectrum, with the differences observed being conserved, in comparison to unactivated complex, after trapping. However, subtle differences in the SID spectra of the activated complex are also observed as a function of trapping time.