Hydrogen/Deuterium Exchange Mass Spectrometry with Top-Down Electron Capture Dissociation for Characterizing Structural Transitions of a 17 kDa Protein
Hydrogen/Deuterium Exchange Mass Spectrometry with Top-Down Electron Capture Dissociation for Characterizing Structural Transitions of a 17 kDa Protein
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DOI：
10.1021/ja904379w
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发表时间：
2009-09-09
影响因子：
15
通讯作者：
Konermann, Lars
中科院分区：
文献类型：
--
作者：
Pan, Jingxi;Han, Jun;Konermann, Lars
酰胺H/D交换（HDX）质谱（MS）广泛用于蛋白质结构研究。传统上，这种技术涉及在D2 O中的蛋白质标记，然后通过酸淬灭、蛋白水解消化和通过HPLC/MS分析肽氘代水平。在开发替代HDX方法中存在极大的兴趣，所述替代HDX方法涉及电喷雾蛋白质离子的自上而下的片段化，而不是依赖于酶促裂解和溶液相分离。最近的一些研究表明，电子捕获解离（ECD）导致气体蛋白质离子的碎片，很少或没有H/D混乱。然而，这种方法在深入的蛋白质构象研究中的成功应用尚未得到证实。目前的工作使用马肌红蛋白作为模型系统，用于评估HDX-MS与自上而下的ECD用于此类实验的适用性。发现ECD可以精确定位受保护的酰胺的位置，对于这种17 kDa蛋白质的平均分辨率小于两个残基。天然全肌红蛋白（hMb）显示出相当大的保护，从交换在其所有的螺旋，而环广泛氘化。在某些螺旋末端可观察到磨损。从hMb中去除辅血红素基团产生脱辅基肌红蛋白（aMb）。hMb和aMb在螺旋A-E中共享几乎相同的HDX保护模式，而螺旋F在aMb中是未折叠的。此外，对于靠近螺旋G的开始、螺旋H的结束和蛋白质的C-末端的一些残基，不稳定是明显的。本文报道的结构变化与抹香鲸肌红蛋白的早期NMR数据基本一致，尽管两个系统之间的微小差异是显而易见的。我们的研究结果表明，自上而下的ECD可获得的结构信息的水平，为小到中等大小的蛋白质大大超过传统的HDX-MS实验，而在同一时间大大减少了不必要的酰胺反交换。
Amide H/D exchange (HDX) mass spectrometry (MS) is widely used for protein structural studies. Traditionally, this technique involves protein labeling in D2O, followed by acid quenching, proteolytic digestion, and analysis of peptide deuteration levels by HPLC/MS. There is great interest in the development of alternative HDX approaches involving the top-down fragmentation of electrosprayed protein ions, instead of relying on enzymatic cleavage and solution-phase separations. A number of recent studies have demonstrated that electron capture dissociation (ECD) results in fragmentation of gaseous protein ions with little or no H/D scrambling. However, the successful application of this approach for in-depth protein conformational studies has not yet been demonstrated. The current work uses horse myoglobin as a model system for assessing the suitability of HDX-MS with top-down ECD for experiments of this kind. It is found that ECD can pinpoint the locations of protected amides with an average resolution of less than two residues for this 17 kDa protein. Native holo-myoglobin (hMb) shows considerable protection from exchange in all of its helices, whereas loops are extensively deuterated. Fraying is observable at some helix termini. Removal of the prosthetic heme group from hMb produces apo-myoglobin (aMb). Both hMb and aMb share virtually the same HDX protection pattern in helices A-E, whereas helix F is unfolded in aMb. In addition, destabilization is evident for some residues close to the beginning of helix G, the end of helix H, and the C-terminus of the protein. The structural changes reported herein are largely consistent with earlier NMR data for sperm whale myoglobin, although small differences between the two systems are evident. Our findings demonstrate that the level of structural information obtainable with top-down ECD for small to medium-sized proteins considerably surpasses that of traditional HDX-MS experiments, while at the same time greatly reducing undesired amide back exchange.