Integrated imaging of individual, mass-selected biomolecules

生物学中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件(如氨基酸和糖)组成的，这些构件以多种不同的方式结合在一起，产生了大量相互作用的成分，这些成分协调着生命所必需的过程，并对疾病的故障负责。然而，这种多样性使得彻底确定生物分子的结构非常困难。如今，几乎所有可用的方法都是对同一分子的多个副本的数据进行平均，模糊了个体差异。因此，结构异质性的许多方面对我们来说实际上是看不见的，我们无法检测和研究它，这就损害了我们理解生命分子机制的能力。解决这个问题的理想方法是以高分辨率逐个检查分子结构。直到最近，我们还缺乏这样做的工具，但由于我们所取得的进展，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质谱学--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相结合。基于我们设计的一种专门的样品处理技术，Prepam MS，我们开发了下一代系统，将来自质谱学的化学成分信息与来自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，这些方法一起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，将包括四个主要组件：质谱仪、将样品从质谱仪转移到成像系统的设备，以及扫描探头和透射式电子显微镜。我们现在寻求的资金是在牛津大学购买并安装这个平台的一个(也是最后一个)组件：扫描探针显微镜，它能够在原子分辨率下进行单分子成像，从而有可能检测单个生物分子之间的结构和组成的细微差异。有了所有的组件，我们将拥有一个完全致力于结构生物分析的集成仪器。该系统将位于卡夫利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。它将被安置在2021年3月开业的一栋新楼里。该平台带来的能力将与研究所内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好处能够得到充分实现，我们计划通过由经验丰富的员工监督的明确定义的访问路线，使牛津和英国的研究小组能够访问该系统。我们平台的能力为许多新的实验应用提供了可能性，使生命科学的一系列探索领域能够取得突破。同时，它将为进一步利用质谱学探测生物结构和功能的方法的进步和改进铺平道路。设想的应用包括结构糖生物学--研究经常添加到细胞中的蛋白质和脂类的糖分子链；膜蛋白--在药物开发和感染中至关重要；以及更广泛地说，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。

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.