Ion Mobility Spectrometry-Tandem Mass Spectrometry for Conformer Selective Struct

描述（由申请人提供）：生物分子在执行其特定功能时所呈现的结构，以及什么变化会停止这些功能，一直是生物学家和化学家的极大兴趣。有许多方法可以探测这些分子在溶液中的结构，每种方法都有各自的优点和缺点。用于电离蛋白质和大（>2 MDa）复合物的技术，甚至来自生物相关的溶液，已经引入质谱作为探测这些分子在气相中的结构的手段。质谱法的优点包括其更高的速度和灵敏度，以及其检查异质混合物和复合物的能力。然而，在气相结构与溶液中观察到的结构之间存在着困难。拟议的工作集中在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。这将通过将能够测量绝对碰撞截面的高分辨率（R~100）静态场IMS漂移管与质谱法和其他表征技术（最值得注意的是电子捕获解离（ECD））相结合来实现。ECD已成为蛋白质测序、探测二级结构和鉴定翻译后修饰位点的有力工具，但绝大多数研究都是从整体上检查生物分子的电荷状态。IMS已经表明，多种构象共存于蛋白质的电荷状态，并且溶剂直接影响构象的分布;关于结构的差异如何影响ECD片段化途径或效率知之甚少。为此，本研究的一个目的是更好地了解离子的横截面如何影响ECD的捕获和碎片化效率。执行此实验需要构建可以容易地耦合到多个质谱仪的IMS漂移室。虽然ECD实验必须在FT/ICR室中进行，但质量分析的时间尺度不适合在所有离子离开漂移管时获得所有离子的质荷比值;相反，将使用IMS耦合到四极（Q-）TOF仪器进行嵌套测量。IMS-Q-TOF还可以用于确定太大而不能通过FT/ICR分析的离子的碰撞截面。目前，只有大的（100 kDa和更大）的复合物的碰撞截面已被测量与行波IMS，低分辨率（R~20）的技术，只提供相对碰撞截面。当与FT/ICR耦合时，漂移管将有助于测量迁移率选择离子的ECD谱，其中绝对碰撞截面是已知的。该过程将最终提高知识的构象离子在真空中表现出，溶剂如何影响这些构象，以及如何潜在地利用这些知识在结构分析和生物分子测序。

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.