A New Approach to Measuring Protein Backbone Protection with High Spatial Resolution Using H/D Exchange and Electron Capture Dissociation
A New Approach to Measuring Protein Backbone Protection with High Spatial Resolution Using H/D Exchange and Electron Capture Dissociation
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DOI：
10.1021/ac401868b
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发表时间：
2013-10-01
影响因子：
7.4
通讯作者：
Kaltashov, Igor A.
中科院分区：
文献类型：
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作者：
Abzalimov, Rinat R.;Bobst, Cedric E.;Kaltashov, Igor A.
空间分辨率不足仍然是氢/氘交换质谱（HDX-MS）最严重的限制之一，特别是当应用于较大的蛋白质（超过30 kDa）。补充蛋白质在溶液中的蛋白质的蛋白水解片段化与在气相中的离子解离已被几个小组成功地使用，以获得近残留水平的分辨率。然而，LC-MS/MS操作模式对数据采集时间范围施加的限制使得在许多情况下难以获得足以可靠分配单个残基处的骨架酰胺保护水平的信噪比。在本工作中，通过消除工作流程中的LC分离步骤，并利用傅里叶变换离子回旋共振质谱仪（FTICR-MS）的高分辨率和动态范围，解除了这种限制。一个残基水平的分辨率被证明为37 kDa的重组蛋白（人血清转铁蛋白的N-叶）的消化片段，使用电子捕获解离作为离子碎片化工具。使用FTICR-MS产生的冗余HDX-MS数据验证了在离子解离之前气相中不存在氢扰乱。通过直接HDX-MS/MS产生的骨架保护模式与已知的蛋白质晶体结构非常一致，但也提供了关于构象动力学的信息，这是静态X射线结构所不能提供的。
Inadequate spatial resolution remains one of the most serious limitations of hydrogen/deuterium exchange-mass spectrometry (HDX-MS), especially when applied to larger proteins (over 30 kDa). Supplementing proteolytic fragmentation of the protein in solution with ion dissociation in the gas phase has been used successfully by several groups to obtain near-residue level resolution. However, the restrictions imposed by the LC-MS/MS mode of operation on the data acquisition time frame makes it difficult in many cases to obtain a signal-to-noise ratio adequate for reliable assignment of the backbone amide protection levels at individual residues. This restriction is lifted in the present work by eliminating the LC separation step from the workflow and taking advantage of the high resolving power and dynamic range of a Fourier transform ion cyclotron resonance-mass spectrometer (FTICR-MS). A residue-level resolution is demonstrated for a peptic fragment of a 37 kDa recombinant protein (N-lobe of human serum transferrin), using electron-capture dissociation as an ion fragmentation tool. The absence of hydrogen scrambling in the gas phase prior to ion dissociation is verified using redundant HDX-MS data generated by FTICR-MS. The backbone protection pattern generated by direct HDX-MS/MS is in excellent agreement with the known crystal structure of the protein but also provides information on conformational dynamics, which is not available from the static X-ray structure.