Ultraviolet Photodissociation Mass Spectrometry for Characterization of Biological Molecules

用于表征生物分子的紫外光解离质谱法

• 批准号: 10389836
• 负责人: Jennifer S. Brodbelt
• 金额: $ 10.04万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389836
• 关键词: Antibiotic Resistance; Area; Binding; Biological; Biomedical Research; C-terminal; Capillary Electrophoresis; Cells; Cellular Membrane; Charge; Chemicals; Collaborations; Complex; Disease; Dissociation; Electrons; Genetic Transcription; Goals; Hybrids; Individual; Ligands; Lipids; Mass Spectrum Analysis; Membrane Proteins; Methods; Microbiology; Modification; Molecular; Molecular Biology; Nucleic Acids; Pattern; Phosphorylation; Post Translational Modification Analysis; Post-Translational Protein Processing; Preparation; Proteins; RNA Polymerase II; Research; Research Support; Sampling; Solvents; Structure; Structure-Activity Relationship; Technology; base; design; disulfide bond; frontier; functional outcomes; innovation; insight; lipid I; macromolecular assembly; mass spectrometer; novel therapeutics; oxidation; pathogenic bacteria; programs; protein complex; protein function; tandem mass spectrometry; ultraviolet

Abstract. Understanding the functions of lipids, proteins and even larger macromolecular assemblies depends on deciphering complex structures of individual molecules as well as decrypting how those molecules interact, often via networks of non-covalent interactions. In order to advance the elucidation of biomolecular organization and functional outcomes, new methods are needed to push the limits of structural insight, providing more detailed holistic chemical information with greater sensitivity. The critical interplay between structure/function is evidenced in numerous biologically-motivated problems, ranging from understanding the ways that pathogenic bacteria develop antibiotic resistance to the design of new drugs that selectively bind and inhibit the functions of protein targets. The ongoing need for even greater chemical insight has motivated my group’s effort to develop innovative mass spectrometry methods to characterize structures of biological molecules in unprecedented detail, especially lipids and proteins which are featured in this proposal. The overarching goal of my research program is to develop state-of- the-art tandem mass spectrometry technologies, particularly highlighting ultraviolet photodissociation (UVPD) and hybrid MS/MS methods, for structural elucidation of lipids, proteins, and protein complexes. These new methods will be showcased for solving challenging problems in three areas. (1) Lipids: (i) profiling lipids of pathogenic bacteria and their signatures of antibiotic resistance, and (ii) structural characterization of unsaturations, oxidations and modifications of lipids that occur during remodeling of cellular membranes. (2) Protein complexes: (i) characterization of protein-ligand complexes, membrane protein complexes, protein/nucleic acid complexes, and macromolecular assemblies, and (ii) advancing capillary electrophoresis for native separations and exploration of the interactome. (3) Post-translational modifications: focusing on decoding the phosphorylation patterns of the C-terminal domain of RNA polymerase II which regulates transcription. These high impact problems are supported via numerous collaborations with microbiology and molecular biology groups who recognize the value of frontier mass spectrometry strategies for elevating biomedical research. This supplement supports acquisition of an ExD cell to enable electron-based activation on existing mass spectrometers and a solvent evaporator to accelerate sample preparation workflows. The ExD cell will support research areas 2 and 3 by allowing electron capture dissociation and charge reduction capabilities for analysis of post-translational modifications and cleavage of disulfide bonds in proteins. The solvent evaporator will facilitate preparation of samples in all three research areas.

抽象的。了解脂质，蛋白质和更大的大分子组件的功能 取决于单个分子的解密复杂结构，并解密了这些结构 分子通常通过非共价相互作用的网络相互作用。为了促进 生物分子组织和功能结果，需要新的方法来推动限制 结构洞察力，以更高的灵敏度提供更详细的整体化学信息。关键 结构/功能之间的相互作用在许多生物学动机问题中证明了 从了解致病细菌对新设计产生抗生素抗性的方式 选择性结合并抑制蛋白质靶标的功能的药物。持续的需求更大 化学见解激发了我小组开发创新的质谱法的努力 以前所未有的细节表征生物分子的结构，尤其是脂质和蛋白质 该提案中有特色。我的研究计划的总体目标是开发 ART串联质谱技术，尤其是突出的紫外线光解离 （UVPD）和杂化MS/MS方法，用于脂质，蛋白质和蛋白质复合物的结构化阐明。 这些新方法将显示用于解决三个领域的挑战问题。 （1）脂质：（i） 致病细菌的脂质及其抗生素耐药性的特征，以及（ii）结构 在重塑过程中发生的不饱和度，氧化和修饰的表征 细胞机制。 （2）蛋白质复合物：（i）蛋白质配体复合物，膜的表征 蛋白质复合物，蛋白/核酸络合物和大分子组件，以及（ii）前进 毛细管电泳，用于天然分离和探索相互作用组。 （3）翻译后 修改：重点是解码RNA的C末端结构域的光磷酸化模式 调节转录的聚合酶II。这些高影响问题通过众多支持 认识到边境质量价值的微生物学和分子生物学组的合作 提升生物医学研究的光谱策略。这种补充支持收购 EXD细胞以实现现有质谱仪和溶剂蒸发器的基于电子的激活 加速样品准备工作流程。 EXD单元将通过允许研究领域2和3 电子捕获解离和电荷还原能力，用于分析翻译后 蛋白质中二硫键的修饰和切割。溶剂蒸发器将有助于 在所有三个研究领域的样品准备。