Ion Mobility Spectrometry-Tandem Mass Spectrometry for Conformer Selective Struct

描述（由申请人提供）：生物学家和化学家都对生物分子在执行其特定功能时所假设的结构以及停止这些功能的变化非常感兴趣。存在许多方法来探测溶液中这些分子的结构，每种方法都有其优缺点。离子化蛋白质和大（bbb20 MDa）复合物的技术，甚至从生物学相关的溶液中，已经引入了质谱法作为探测这些分子在气相中的结构的手段。质谱法的优点包括更快的速度和灵敏度，以及检测非均质混合物和复合物的能力。然而，在将气相结构与在溶液中观察到的结构联系起来时，出现了困难。提出的工作重点是更好地理解从溶液到气相的过渡如何影响生物大分子的结构，特别是，目前许多气相技术无法区分的记忆离子可能具有其溶剂。这将通过将高分辨率（R~100）静态场IMS漂移管（能够测量绝对碰撞截面）与质谱和其他表征技术（最值得注意的是电子捕获解离（ECD））相结合来实现。ECD已经成为蛋白质测序、探测二级结构和识别翻译后修饰位点的有力工具，但绝大多数研究都是将生物分子的电荷状态作为一个整体进行检查。IMS已经表明，多种构象共存于蛋白质的一个电荷状态，溶剂直接影响构象的分布；对于结构差异如何影响ECD裂解途径或效率，我们知之甚少。为此，本研究的目的之一是更好地了解离子的横截面如何影响ECD的捕获和破碎效率。进行这个实验需要建立一个IMS漂移单元，它可以很容易地与几个质谱仪耦合。虽然ECD实验必须在FT/ICR细胞中进行，但质量分析的时间尺度不适合获得所有离子离开漂移管时的质量电荷比值；取而代之的是，嵌套测量将使用IMS耦合到四极（Q-） TOF仪器。IMS-Q-TOF还可以用于确定大到无法用FT/ICR分析的离子的碰撞截面。目前，唯一的大型（100 kDa及更大）配合物的碰撞截面是用行波IMS测量的，这是一种低分辨率（R~20）的技术，只能提供相对碰撞截面。当与FT/ICR耦合时，漂移管将有助于测量已知绝对碰撞截面的迁移率选定离子的ECD光谱。该过程将最终提高在真空中表现的构象的知识，溶剂如何影响这些构象，以及如何在结构分析和生物分子测序中潜在地应用这些知识。

DESCRIPTION (provided by applicant): The structures assumed by biological molecules as they perform their specific functions, as well as what changes cease those functions, have been of great interest to biologists and chemists alike. Many methods exist for probing the structures of these molecules in solution, each with their strengths and weaknesses. Techniques for ionizing proteins and large (>2 MDa) complexes, even from biologically relevant solutions, have introduced mass spectrometry as a means of probing the structures of these molecules in the gas phase. Advantages of mass spectrometry include its greater speed and sensitivity, as well as its ability to examine heterogeneous mixtures and complexes. However, difficulties arise in relating gas phase structures to those observed in solution. The proposed work is focused at better understanding how the transition from solution to gas phase influences the structures of biological macromolecules, specifically, what memory ions that are currently indistinguishable by many gas phase techniques might have of their solvent. This will be achieved by coupling a high resolution (R~100) static-field IMS drift tube, capable of measuring absolute collision cross sections, with mass spectrometry and other characterization techniques, most notably electron capture dissociation (ECD). ECD has become a powerful tool in protein sequencing, probing secondary structure, and identifying sites of post-translational modifications, but the vast majority of studies have examined a charge state of a biomolecule as a whole. IMS has shown that multiple conformations coexist across a charge state of a protein, and that solvent directly influences the distribution of conformations; little is known regarding how differences in structure affect either ECD fragmentation pathways or efficiencies. To this end, one aim of this study is to better understand how the cross section of an ion affects both the capture and fragmentation efficiencies of ECD. Performing this experiment requires building an IMS drift cell that can couple easily to several mass spectrometers. While ECD experiments must be performed in the FT/ICR cell, the timescale of the mass analysis is not amenable to obtaining mass-to-charge values for all ions as they exit the drift tube; instead, nested measurements will be made using IMS coupled to a quadropole (Q-) TOF instrument. The IMS-Q-TOF can also be employed for determining collision cross sections for ions too large to be analyzed by FT/ICR. Currently, the only collision cross sections for large (100 kDa and greater) complexes have been measured with the travelling wave IMS, a low-resolution (R~20) technique that only provides relative collision cross sections. When coupled to the FT/ICR, the drift tube will facilitate the measurement of ECD spectra of mobility selected ions for which the absolute collision cross sections are known. The process will ultimately improve knowledge of the conformations ions exhibit in vacuum, how solvent influences those conformers, and how to potentially employ this knowledge in both structural analysis and biomolecular sequencing. PUBLIC HEALTH RELEVANCE: The gas phase provides an interesting regime in which to probe biological macromolecules and complexes too large and/or complicated to be probed by solution phase methods. Relationships between the gas phase conformations of these molecules and the solvent from which they came could provide complimentary information to that obtained with methods such as nuclear magnetic resonance and X-ray crystallography. To this end, ion mobility spectrometry will be employed to obtain absolute cross sectional measurements of these molecules, as well as to select specific conformational subsets for characterization with mass spectrometric techniques, initially electron capture dissociation, with a focus on biologically relevant systems, including (but not limited to) the Barnase/Barstar complex, the sigma 54 (Ntr-C4-RC) complex, and the anthrax toxin complex.