Structure, Function and Regulation of the Proteome

蛋白质组的结构、功能和调控

• 批准号: 10401900
• 负责人: JOSHUA J COON
• 金额: $ 87.78万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-05 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10401900
• 关键词: Achievement; Attention; Biomedical Research; Cell physiology; Chemicals; Cryoelectron Microscopy; Development; Developmental Biology; Dissociation; Electron Microscopy; Electron Transport; Gases; Ice; Image; Investigation; Ions; Laboratories; Mass Spectrum Analysis; Measurement; Metabolism; Methodology; Methods; Mission; Molecular; Monitor; Names; Phase; Preparation; Proteins; Proteome; Proteomics; Reaction; Regulation; Resolution; Sampling; Shotguns; Speed; Structure; Surface; Systems Biology; Techniques; Technology; astrophysics; biological systems; cryogenics; density; innovation; insight; instrumentation; lipidome; macromolecule; mass spectrometer; metabolome; nanometer; new technology; novel; particle; programs; protein complex; protein structure; structural biology

PROJECT SUMMARY My laboratory innovates mass spectrometry (MS) technology to accelerate the pace, depth, and accuracy of proteome analysis and applies these technologies to globally access proteome structure, function, and regulation. Signature achievements include the development of electron-transfer dissociation (ETD), the GC- Orbitrap mass spectrometer, novel protein quantification technologies, and the targeted technique parallel reaction monitoring – to name a few. Having established our reputation in proteomics instrumentation, our mission came to include the investigation of the metabolome and lipidome. The rationale is simple: fully understanding a biological system requires knowing the interplay between molecular classes. We have demonstrated the capability of these technologies to propel numerous projects in the fields of metabolism, developmental, and systems biology. Building on my program’s expertise in the analysis of disassembled molecular machinery (i.e., shotgun proteomics), we have turned our attention to the machine as a whole, i.e., protein structure. By combining technologies from MS and cryogenic electron microscopy (cryo-EM) our aim is to maximize the information garnered from a single sample to provide insight into protein structure with unprecedented speed and simplicity. That is, we compound the orthogonal information offered by MS measurements of chemical makeup with atomic- level information afforded by cryo-EM imaging. Further, drawing on concepts from astrophysics already resonant with MS, we anticipate that our highly creative method will overcome pervasive bottlenecks in cryo-EM sample preparation that often limit image resolution. To that end, we have adapted a mass spectrometer to purify and land native protein complexes on cryogenically cooled TEM grids, which are then coated with amorphous ice in vacuo. The resulting grid would represent the ideal cryo-EM sample – a high density of particles situated in random orientations in the same focal plane and covered with only a few nanometers of ice. We plan to achieve even greater structural insight through the incorporation of gas-phase dissociation techniques, which partition the macromolecule into its subcomponents. In particular we propose the addition of surface-induced dissociation (SID) and activated-ion ETD (AI-ETD) to the mass spectrometer used for grid preparation. In short, this technology has genuine potential to create a new paradigm of proteome-scale structural biology.

项目摘要 我的实验室创新了质谱（MS）技术，以加快研究的步伐、深度和准确性。 蛋白质组分析，并应用这些技术在全球范围内访问蛋白质组结构，功能， 调控标志性成就包括电子转移解离（ETD）的发展，GC- 轨道阱质谱仪、新型蛋白质定量技术和靶向技术并行 反应监测-仅举几例。我们在蛋白质组学仪器方面建立了声誉， 使命包括代谢组和脂质组的研究。理由很简单：充分 了解生物系统需要了解分子类别之间的相互作用。我们有 展示了这些技术推动新陈代谢领域众多项目的能力， 发展和系统生物学。 基于我的程序在分析分解分子机器方面的专业知识（即，猎枪 蛋白质组学），我们已经将注意力转向整个机器，即，蛋白质结构通过组合 我们的目标是最大限度地利用MS和低温电子显微镜（cryo-EM）技术， 从单个样品中获得，以前所未有的速度和简单性深入了解蛋白质结构。 也就是说，我们将化学组成的MS测量所提供的正交信息与原子- 冷冻电磁成像提供的水平信息。此外，借鉴天体物理学的概念， 有了MS，我们预计我们高度创造性的方法将克服冷冻EM样品中普遍存在的瓶颈 这通常会限制图像分辨率。为此，我们采用了质谱仪来纯化和 将天然蛋白质复合物降落在低温冷却的TEM网格上，然后用无定形冰覆盖， 真空。由此产生的网格将代表理想的冷冻EM样品-位于样品中的高密度颗粒。 在同一个焦平面上的随机取向，覆盖着只有几纳米的冰。我们计划实现 甚至更大的结构洞察力，通过结合气相解离技术，其中分区 将大分子分解成它的子成分。特别地，我们建议增加表面诱导解离 (SID)和活化离子ETD（AI-ETD）到用于网格制备的质谱仪。总之这 技术具有创造蛋白质组规模结构生物学新范例的真正潜力。