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

An increasing number of studies on biomolecular function indirectly combine mass spectrometry (MS) with imaging techniques such as cryo electron microscopy (cryo-EM). This approach allows information on the homogeneity, stoichiometry, shape, and interactions of native protein complexes to be obtained, complementary to high-resolution protein structures. We have recently demonstrated TEM sample preparation via native electrospray ion-beam deposition (ES-IBD) as a direct link between native MS and cryo-EM. This workflow forms a potential new route to the reliable preparation of homogeneous cryo-EM samples and a better understanding of the relation between native solution-phase and native-like gas-phase structures. However, many aspects of the workflow need to be understood and optimized to obtain performance comparable to that of state-of-the-art cryo-EM. Here, we expand on the previous discussion of key factors by probing the effects of substrate type and deposition energy. We present and discuss micrographs from native ES-IBD samples with amorphous carbon, graphene, and graphene oxide, as well as landing energies in the range between 2 and 150 eV per charge

A preparative mass spectrometer to deposit intact large native protein complexes

Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study the structure and reactivity of molecules from small metal clusters to large protein assemblies. It brings molecules gently into the gas phase, where they can be accurately manipulated and purified, followed by controlled deposition onto various substrates. In combination with imaging techniques, direct structural information on well-defined molecules can be obtained, which is essential to test and interpret results from indirect mass spectrometry techniques. To date, ion-beam deposition experiments are limited to a small number of custom instruments worldwide, and there are no commercial alternatives. Here we present a module that adds ion-beam deposition capabilities to a popular commercial MS platform (Thermo Scientific Q Exactive UHMR mass spectrometer). This combination significantly reduces the overhead associated with custom instruments, while benefiting from established high performance and reliability. We present current performance characteristics including beam intensity, landing-energy control, and deposition spot size for a broad range of molecules. In combination with atomic force microscopy (AFM) and transmission electron microscopy (TEM), we distinguish near-native from unfolded proteins and show retention of the native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify the activity of a noncovalent protein complex after deposition on a dry surface. Together, these results not only indicate a great potential of ES-IBD for applications in structural biology, but also outline the challenges that need to be solved for it to reach its full potential

Cryo-EM of soft-landed β-galactosidase: gas-phase and native structures are remarkably similar

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single particle electron cryomicroscopy (cryo-EM) down to 2.6 ˚A resolution, enabled by soft-landing of mass selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in-situ ice coating. We find that large parts of the secondary and tertiary structure are retained from solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native MS and cryo-EM

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single-particle cryo–electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM

Preparative mass spectrometry instrumentation for large native protein deposition

Electrospray-ion-beam deposition (ES-IBD) is a preparative mass spectrometry technique which produces highly purified samples of non-volatile molecules adsorbed on a surface. An ES-IBD mass spectrometer (MS) ionises the analyte, transfers it into the gas phase, removes contaminations with a mass filter in vacuum and deposits it with a controlled landing energy onto a substrate. ES-IBD coupled to microscopy can elucidate the structure of biomolecules and their assemblies. In conjunction with scanning tunnelling microscopy, glycan connectivity of oligosaccharides was revealed. For larger molecules, such as native protein complexes, efforts are underway to determine their structure and conformation by coupling ES-IBD to cryo-electron microscopy or low-energy electron holography. However, there are no instruments available dedicated to depositing large, native protein complexes controlled and efficiently. In this thesis, I have modified a commercial MS designed for the analysis of large, native protein complexes (Thermo ScientificTM Q ExactiveTM UHMR) for deposition. The modified instrument has adequate beam intensity and good landing energy control. In conjunction with electron microscopy, it was shown that the native shape of protein assemblies is retained after dehydration and deposition. I used an enzymatic assay to quantify the activity of the non-covalent protein complex alcohol dehydrogenase after deposition on a dry surface. Moreover, I developed another instrument for mass- and mobility-filtered deposition of both small and large molecules. Its atmosphere-pressure interface, the ion funnel, is optimised for high transmission and it has a continuous parallel flow ion mobility filter to enable conformation-selected deposition. Radio-frequency power switches with a flexible operation frequency permit to transmit any ion with a mass-to-charge range > 50 Th, in particular heavy ions. The basis for the development of the second MS was the detailed investigation of the transport processes within an ion funnel. I formulate a 4-step ion funnel transmission model underpinned by transmission measurements of a conventional ion funnel and ion trajectory simulations taking into account the gas flow field

Portable Analyzer for On-Site Determination of Dissolved Organic Carbon — Development and Field Testing

Dissolved organic carbon (DOC) is a sum parameter that is frequently used in water analytics. Highly resolved and accurate DOC data are necessary, for instance, for water quality monitoring and for the evaluation of the efficiency of treatment processes. The conventional DOC determination methods consist of on-site sampling and subsequent analysis in a stationary device in a laboratory. However, especially in regions where no or only poorly equipped laboratories are available, this method bears the risk of getting erroneous results. For this reason, the objective of the present study was to set up a reliable and portable DOC analyzer for on-site analysis. The presented DOC system is equipped with an electrolysis-based decomposition cell with boron-doped diamond electrodes (BDD) that oxidizes the organic compounds to carbon dioxide. Within this study, the influence of different electrode materials and the composition of the applied electrolytes on the DOC decomposition in an undivided electrolytic cell were systematically investigated. Furthermore, some technical aspects of the portable prototype are discussed. After a detailed validation, the prototype was used in an ongoing monitoring program in Northern India. The limit of detection is 0.1 mg L−1 C with a relative standard deviation of 2.3% in a linear range up to 1000 mg L C−1. The key features of the portable DOC analyzer are: No need for ultra-pure gases, catalysts or burning technology, an analyzing time per sample below 5 min, and a reliable on-site DOC determination

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

Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas phase and solution phase protein structures