Structure, Function and Regulation of the Proteome

项目总结 我的实验室创新了质谱学(MS)技术，以加快研究的速度、深度和准确性 蛋白质组分析并应用这些技术来全球访问蛋白质组结构、功能和 监管。标志性的成就包括电子转移解离(ETD)的发展，GC- Orbitrap质谱计、蛋白质定量新技术和靶向技术并行 反应监测--仅举几例。在建立了我们在蛋白质组学仪器方面的声誉后，我们的 任务开始包括对代谢组和脂质体的研究。理由很简单：完全 了解一个生物系统需要知道分子类之间的相互作用。我们有 展示了这些技术推动新陈代谢领域的众多项目的能力， 发展生物学和系统生物学。 基于我的程序在分析分解的分子机械(即猎枪)方面的专业知识 蛋白质组学)，我们已经将注意力转向机器作为一个整体，即蛋白质结构。通过组合 来自MS和低温电子显微镜的技术我们的目标是最大限度地利用 从单个样本获取，以前所未有的速度和简单性提供对蛋白质结构的洞察。 也就是说，我们将化学成分的MS测量提供的正交信息与原子- 低温电磁成像提供的水平信息。此外，借鉴已经引起共鸣的天体物理学的概念 有了MS，我们预计我们高度创新的方法将克服冷冻-EM样品中普遍存在的瓶颈 通常会限制图像分辨率的准备工作。为此，我们改装了质谱仪来提纯和 将天然蛋白质复合体放在低温冷却的电子显微镜网格上，然后在网格上覆盖无定形冰 真空吸尘器。由此产生的网格将代表理想的低温EM样本--位于 在同一焦平面上随机定向，并且只覆盖了几个纳米的冰。我们计划实现 通过结合气相分离技术获得更大的结构洞察力， 将大分子分解成其亚组分。特别地，我们建议增加表面诱导解离。 (SID)和激活离子ETD(AI-ETD)到用于网格制备的质谱计。简而言之，这就是 技术具有创造蛋白质组级结构生物学新范式的真正潜力。

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.