A custom deposition stage, controlled using a home-built software, was added to a commercial mass spectrometer (Thermo Scientific Q Exactive UHMR). The modified instrument allows to produce intense ion-beams of native proteins and protein complexes for deposition onto TEM grids and graphite samples. Beam intensity, total beam energy, and beam energy distribution were characterized and optimized. Mass-selective, clean, protein samples with consistent quality can typically be prepared in less than 30 minutes. Soft-landed samples of protein complexes in a mass range from 150 to 800 kDa were imaged using atomic force microscopy (AFM), negative stain TEM, and cryo-EM. For cryo-EM, 2 nm amorphous carbon films were identified as ideal substrate due to high contrast and suppression of thermal diffusion. The results show that protein shapes remain in line with known native protein structures when limiting landing energies to below 10 eV per charge. 2D classes and 3D EM density maps from soft-landed samples show that proteins remain folded and subunits in protein complexes remain attached, confirming retention of near-native structures of large protein complexes in the gas-phase. The resolution did not allow for determination of secondary structure. A series of control experiments suggest that preparation of protein solutions for mass spectrometry, microscope settings, preferred orientation, beam-induced motion, are not limiting. Instead, our results indicate that heterogeneity in the secondary and tertiary structure, introduced by dehydration, radiation damage, landing, or surface interactions, limits the amount of information that can be obtained by averaging techniques.
A cryogenically cooled landing stage was implemented to control temperature and hydration throughout the workflow. It forms the basis for variations of the workflow that include controlled rehydration, which is essential to retain or regain native solution phase structures. Unfortunately, COVID-19 related delays in the delivery of essential parts delayed completion until shortly after the end of the project.
The results were presented at the conference for Isolated Biomolecules and Biomolecular Interactions (IBBI) in 2022. Further disseminations shortly after the end of the project include talks at the International Mass Spectrometry Conference (IMSC) 2022, the Faraday discussion on “Challenges in biological cryo electron microscopy” in 2022, and the workshop “Frontiers in Native Mass Spectrometry and Single-Molecule Imaging”, co-organized by the researcher. A preprint focusing on general instrument performance and applications as posted to arxiv and submitted to a high-impact journal. It is, as of July 2022, in minor revision. A publication on the specific use for cryo-EM sample preparation has been accepted by PNAS Nexus. A third paper focusing on the choice of the substrate and surface induced dissociation has been accepted as part of the Faraday discussion mentioned above. Thermo Fisher Scientific and the University of Oxford have filed a joint patent application concerning variations of the workflow beyond the action. The close collaboration with Thermo Fisher Scientific on this project resulted in a unique opportunity for the fellow to join the company while being able to complete construction and characterization of the cryo landing stage at the University of Oxford.
