Integrated imaging of individual, mass-selected biomolecules

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.

Understanding the structure, dynamics, and function of proteins and other biopolymers is difficult due to their extensive heterogeneity, which arises from alternative sequences, modifications and oligomerisation. This diversity controls how proteins and other biopolymers function and interact. However, virtually all methods today average data from multiple copies of a molecule, blinding us to this critical variability. Our inability to detect and investigate heterogeneity compromises our understanding of the molecular mechanisms of life.

The ideal way to overcome this is to characterise molecules individually at high resolution. Recent developments have turned this ambitious idea into a realistic prospect, promising a revolution for the structural characterisation of biomolecules. Our solution combines mass spectrometry (MS) and single-molecule imaging. It builds on preparative MS, a technique we pioneered that transfers molecules of interest, intact, into the vacuum of the mass spectrometer; selects molecules in specific states; and then deposits them onto a surface held at ultra-high vacuum, for transfer to a microscope.

Our system, the first like it in the world, links MS data with detailed structural information from high-resolution imaging by transmission electron microscopy and scanning probe microscopy. We seek funding to acquire and integrate the final component into the platform in Oxford: the scanning probe microscope. With all components then in place, we will have an integrated instrument dedicated to biological analyses.

The instrument will enable new experimental applications and breakthroughs across the life sciences, for which we will make it accessible to researchers across Oxford and the UK, while paving the way for further methodological advances and refinement. Envisioned applications include glycobiology, membrane proteins, and our general understanding of the roles of protein modification and heterogeneity in the processes of life.