A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes

Abstract

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

“…The total energy distribution of the ion beam has a full width at half maximum of 1 eV per charge. 34 Thus, most proteins land with an energy in the range from 1.5 to 2.5 eV per charge. We do not see any distribution in particle quality corresponding to this deposition energy range, but cannot exclude that even lower deposition energies are required to increase sample quality.…”

“…1 and described in detail elsewhere. 33,34 Protein solutions were prepared using a standard native MS workflow described below. Protein assemblies were transferred into the gas phase via native electrospray ionization using gold coated borosilicate capillaries, prepared in house.…”

“…We have recently developed a native mass spectrometer with ion-beam deposition capability for the preparation of mass-selective and ice-free samples for imaging under cryo conditions using native ES-IBD. 33,34 Our setup is based on a commercial, high-resolution, tandem mass spectrometer (Thermo Scientific™ Q Exactive™ UHMR mass spectrometer), to which we have added a custom deposition stage. With this instrument, we have shown that even the non-covalently bound tetramer of alcohol dehydrogenase (ADH) preserves its enzymatic activity after deposition onto a dry carbon substrate.…”

“…With this instrument, we have shown that even the non-covalently bound tetramer of alcohol dehydrogenase (ADH) preserves its enzymatic activity after deposition onto a dry carbon substrate. 34…”

Matrix-Landing Mass Spectrometry for Electron Microscopy Imaging of Native Protein Complexes

“…For several decades, practitioners of mass spectrometry (MS) have sought to use MS as a preparative tool. − Early works by Robinson et al demonstrated the potential to land protein complex cations onto transmission electron microscopy (TEM) grids for imaging after negative staining. However, the negatively stained images lacked detail, and it is therefore difficult to judge how well the underlying structures were preserved. , More recent work by Rauschenbach et al directly imaged protein complex ions without staining, soft landing them at room temperature and imaging at liquid nitrogen temperature. − In those experiments, however, class averaging and 3D reconstruction of the landed protein complexes demonstrated no internal detail suggesting the structures have been perturbed in some way.…”