Ion Mobility Spectrometry-Tandem Mass Spectrometry for Conformer Selective Struct

构象选择性结构的离子淌度-串联质谱分析

• 批准号: 7912831
• 负责人: Samuel Isiah Merenbloom
• 金额: $ 4.56万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912831
• 关键词: Affect; Amino Acid Sequence; Bacillus amyloliquefaciens barstar protein; Bacillus amyloliquefaciens ribonuclease; Biological; Cells; Charge; Complex; Complex Mixtures; Coupled; Coupling; Dissociation; Electrons; Exhibits; Gases; Ions; Knowledge; Mass Spectrum Analysis; Measurement; Measures; Memory; Methods; Molecular Conformation; Nuclear Magnetic Resonance; Pathway interactions; Peptide Sequence Determination; Phase; Post-Translational Modification Site; Process; Proteins; RNA polymerase sigma 54; Relative (related person); Resolution; Solutions; Solvents; Spectrometry; Speed; Structure; System; Techniques; Travel; Tube; Vacuum; Work; X-Ray Crystallography; anthrax toxin; conformer; improved; instrument; interest; ion mobility; macromolecule; mass spectrometer; public health relevance; research study; tandem mass spectrometry; tool

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.

描述(申请人提供)：生物学家和化学家都非常感兴趣的是，生物分子在执行其特定功能时所采用的结构，以及什么变化终止了这些功能。有许多方法可以探测这些分子在溶液中的结构，每种方法都有它们的优点和缺点。用于电离蛋白质和大的(&gt；2丙二醛)复合体的技术，甚至从生物相关的溶液中，已经引入了质谱学作为探测这些分子在气相中的结构的一种手段。质谱学的优势包括它更快的速度和灵敏度，以及它检测非均相混合物和络合物的能力。然而，将气相结构与溶液中观察到的气相结构联系起来存在困难。拟议的工作重点是更好地理解从溶液到气相的转变如何影响生物大分子结构，特别是目前许多气相技术无法区分的记忆离子可能具有的溶剂。这将通过将一个能够测量绝对碰撞截面的高分辨率(R~100)静电场IMS漂移管与质谱学和其他表征技术相结合来实现，最著名的是电子俘获解离(ECD)。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强大工具，但绝大多数研究都是从整体上考察生物分子的电荷状态。IMS已经表明，在蛋白质的电荷状态下，多种构象共存，而溶剂直接影响构象的分布；关于结构差异如何影响ECD碎裂途径或效率，人们知之甚少。为此，这项研究的目的之一是更好地了解离子的横截面如何影响ECD的捕获和碎裂效率。进行这项实验需要建立一个IMS漂移池，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池中进行，但质量分析的时间刻度不能获得所有离子离开漂移管时的质荷比值；相反，将使用耦合到四极(Q-)TOF仪器的IMS进行嵌套测量。IMS-Q-TOF还可以用来确定太大而无法用FT/ICR分析的离子的碰撞截面。目前，仅有的大型(100 kDa或更大)络合物的碰撞截面是用行波IMS测量的，行波IMS是一种只提供相对碰撞截面的低分辨率(R~20)技术。当与FT/ICR耦合时，漂移管将有助于测量迁移率选定离子的ECD谱，这些离子的绝对碰撞截面已知。这一过程最终将改善离子在真空中表现出的构象的知识，溶剂如何影响这些构象，以及如何潜在地将这些知识应用于结构分析和生物分子测序。 与公共健康相关：气相提供了一种有趣的机制，可以用来探测太大和/或太复杂的生物大分子和复合体，而不是用溶液相方法来探测。这些分子的气相构象与它们所来自的溶剂之间的关系可以为核磁共振和X射线结晶学等方法所获得的信息提供补充。为此，将使用离子迁移率光谱来获得这些分子的绝对截面测量，以及选择特定的构象亚集用于用质谱学技术进行表征，首先是电子俘获解离，重点是生物相关的系统，包括(但不限于)Barnase/Barstar复合体、sigma 54(NTR-C4-RC)复合体和炭疽毒素复合体。