Integrated imaging of individual, mass-selected biomolecules

单个、大量选择的生物分子的集成成像

• 批准号: BB/V019694/1
• 负责人: Stephan Rauschenbach
• 金额: $ 84.37万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV019694%2F1
• 关键词: Integrated; imaging; individual; mass; selected

A major challenge in biology is to understand the function, structure and dynamics of proteins and other biomolecules. These biomolecules are built from a small number of building blocks (such as amino acids and sugars), which are combined in a multitude of different ways, creating a vast diversity of interacting components that orchestrate the processes that are necessary for life and responsible for malfunction in disease.This diversity, however, makes it very difficult to thoroughly determine the structure of biomolecules. Virtually all methods available today average data from multiple copies of a molecule, blurring individual differences. As a result, many aspects of structural heterogeneity are effectively invisible to us, and our inability to detect and investigate it compromises our ability to understand the molecular mechanisms of life. The ideal way to overcome this problem is to examine the structure of molecules, one by one and at high resolution. Until recently we lacked the tools to do this, but due to advances we have made, this is now a realistic prospect, promising a revolution for the structural characterisation of biomolecules. Our solution is to combine mass spectrometry, the highest resolution way of separating and measuring the mass of proteins, with atomic-resolution imaging of single molecules. Building on a specialised sample handling technique we devised, prepMS, we have developed a next-generation system that links chemical composition information from mass spectrometry with detailed structural information from high-resolution imaging. We use complementary imaging approaches, electron microscopy and scanning probe microscopy, which together enable us to gather high-resolution and three-dimensional structural data. Our platform, the first of its kind in the world, will involve four main components: a mass spectrometer, an apparatus for transferring samples from the mass spectrometer to the imaging systems, and both scanning probe and transmission electron microscopes. The funding we now seek is to purchase and install one, single (and final) component of this platform at the University of Oxford: a scanning probe microscope, which is capable of single molecule imaging at atomic resolution, making it possible to detect subtle differences in structure and composition among individual biomolecules. With all the components then in place, we will have an integrated instrument entirely dedicated to structural biological analyses.The system will be located in the Kavli Institute for Nanoscience Discovery, which is being set up to enable frontier physical sciences methods to deliver new insights at the frontiers of biology. It will be housed in a new building opening in March 2021. The capabilities the platform brings will have relevance to the many researchers who study biomolecular structure-in the institute and more widely. To ensure the system's considerable benefits can be fully realised, we plan to make it accessible to research groups across Oxford and the UK through well-defined access routes overseen by experienced staff.Our platform's capabilities open up the possibility of many new experimental applications, enabling breakthroughs in a range of areas of exploration across the life sciences. Meanwhile, it will pave the way for further methodological advances and refinement in the use of mass spectrometry to probe biological structure and function. Envisioned applications include the vastly understudied field of structural glycobiology - the study of chains of sugar molecules that are frequently added to proteins and lipids in cells; membrane proteins - which are critically important in drug development and in infection; and, more generally, our fundamental understanding of the roles of protein modification and biomolecular heterogeneity in the processes of life.

生物学的一个主要挑战是了解蛋白质和其他生物分子的功能，结构和动力学。这些生物分子是由少量的构建单元（如氨基酸和糖）组成的，它们以多种不同的方式组合在一起，产生了各种各样的相互作用的成分，这些成分协调着生命所必需的过程，并导致疾病的功能障碍。然而，这种多样性使得彻底确定生物分子的结构非常困难。事实上，今天所有可用的方法都是从一个分子的多个拷贝中平均数据，模糊了个体差异。因此，结构异质性的许多方面实际上对我们来说是不可见的，我们无法检测和研究它，这损害了我们理解生命分子机制的能力。克服这个问题的理想方法是逐个地以高分辨率检查分子的结构。直到最近，我们还缺乏这样做的工具，但由于我们已经取得的进展，这现在是一个现实的前景，有望为生物分子的结构表征带来一场革命。我们的解决方案是将联合收割机质谱（分离和测量蛋白质质量的最高分辨率方法）与单分子的原子分辨率成像结合起来。基于我们设计的专业样品处理技术prepMS，我们开发了一种新一代系统，该系统将质谱分析的化学成分信息与高分辨率成像的详细结构信息联系起来。我们使用互补的成像方法，电子显微镜和扫描探针显微镜，它们共同使我们能够收集高分辨率和三维结构数据。我们的平台是世界上第一个此类平台，将包括四个主要组成部分：质谱仪，将样品从质谱仪转移到成像系统的装置，以及扫描探针和透射电子显微镜。我们现在寻求的资金是在牛津大学购买和安装该平台的一个单一（也是最后一个）组件：扫描探针显微镜，它能够以原子分辨率进行单分子成像，从而可以检测单个生物分子之间结构和组成的细微差异。随着所有组件的到位，我们将拥有一个完全致力于结构生物学分析的集成仪器。该系统将位于Kavli纳米科学发现研究所，该研究所正在建立，以使前沿物理科学方法能够在生物学前沿提供新的见解。它将位于2021年3月开放的新大楼内。该平台带来的能力将与研究所和更广泛地研究生物分子结构的许多研究人员相关。为了确保该系统的巨大优势能够得到充分发挥，我们计划通过经验丰富的工作人员监督的明确的访问路线，让牛津和英国的研究小组能够访问该系统。我们平台的功能为许多新的实验应用开辟了可能性，使生命科学的一系列探索领域取得突破。同时，它将为使用质谱法探测生物结构和功能的进一步方法进步和改进铺平道路。设想的应用包括结构糖生物学的研究领域-糖分子链的研究，这些糖分子链经常被添加到细胞中的蛋白质和脂质中;膜蛋白-这在药物开发和感染中至关重要;以及更普遍地，我们对蛋白质修饰和生物分子异质性在生命过程中的作用的基本理解。

项目成果

Cryo-EM samples of gas-phase purified protein assemblies using native electrospray ion-beam deposition.

• DOI: 10.1039/d2fd00065b
• 发表时间: 2022-11-08
• 期刊: FARADAY DISCUSSIONS
• 影响因子: 3.4
• 作者: Esser, Tim K.;Bohning, Jan;Fremdling, Paul;Bharat, Tanmay;Gault, Joseph;Rauschenbach, Stephan
• 通讯作者: Rauschenbach, Stephan

A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes.

• DOI: 10.1021/acsnano.2c04831
• 发表时间: 2022-09-27
• 期刊: ACS NANO
• 影响因子: 17.1
• 作者: Fremdling, Paul;Esser, Tim K.;Saha, Bodhisattwa;Makarov, Alexander A.;Fort, Kyle L.;Reinhardt-Szyba, Maria;Gault, Joseph;Rauschenbach, Stephan
• 通讯作者: Rauschenbach, Stephan

Pushing the limits in single particle cryo-EM: general discussion.

突破单粒子冷冻电镜的极限：一般讨论。

• DOI: 10.1039/d2fd90063g
• 发表时间: 2022
• 期刊: Faraday discussions
• 影响因子: 3.4
• 作者: Bakker SE

Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition

通过自然电喷雾离子束沉积进行质量选择性和无冰冷冻电镜蛋白质样品制备

• DOI: 10.1101/2021.10.18.464782
• 发表时间: 2021
• 作者: Esser T