Integrated imaging of individual, mass-selected biomolecules

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry


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.

Technical Summary

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.
Description Key goal was implementation of the imaging pipeline, which has been achieved (with the exception of the direct coupling for cryo deposition).

This means now the high resolution imaging of small and large bio molecules by STM and cryoEM is possible.

Our first publications in this field are received with great interest.
Exploitation Route Followup funding has already been obtained. There will be at least three projects active on the instrument in the next year, others likely to come in.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

Description BBSRC grant: Mapping Protein Glycosylation by High-Resolution Single Molecule Imaging
Amount £538,533 (GBP)
Funding ID BB/W017024/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2023 
End 09/2026
Description Imaging Interaction of Proteins and Drugs at the Single Molecule Level.
Amount £212,000 (GBP)
Organisation Vertex Pharmaceuticals 
Sector Private
Country United States
Start 02/2023 
End 02/2027
Title Electrospray Ion Beam Deposition of molecules for SPM imaging based on a UHMR Orbitrap instrument 
Description This grant resulted in the implementation of the first commercial platform, high resolution mass spectrometer modified to be used as a molecular deposition instrument for ultra pure molecular coatings in vacuum. 
Type Of Material Improvements to research infrastructure 
Year Produced 2022 
Provided To Others? Yes  
Impact Researchers in the field develop similar instrumentation or attempt to purchase them Commercial vendors consider developing user instruments based on the research. 
Description Collaboration with Thermo Fisher Scientific (TFS): preparative mass spectrometer based on commercial orbitrap platform. 
Organisation Thermo Fisher Scientific
Department Thermo Fisher Scientific, Germany
Country Germany 
Sector Private 
PI Contribution Our contribution: - modification of the instrument - characterisation of the instrument - scientific work on protein structure - fundamental work on particle surface collision
Collaborator Contribution TFS contribution: - permanent instrument loan - scientific and technical support including custom parts
Impact First publications start to appear. [1] \n A preparative mass spectrometer to deposit intact large native protein complexes 2022-03-11 | Journal article DOI: 10.48550/ARXIV.2203.04671 [2] Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition bioRxiv 2021 | Other DOI: 10.1101/2021.10.18.464782 EID: 2-s2.0-85119485606 Part of ISSN: 26928205 - Joint IP has been filed with university. - A joint patent has been submitted.
Start Year 2019