Direct single molecule imaging on a modified Q Exactive UHMR with electron holography capability
Albert Konijnenberg1, Josh Gilbert2, Kyle Fort3, Alexander Makarov3, Alan Bahm2
1 Thermo Fisher Scientific, Achtseweg Noord 5, 5651 GG Eindhoven, the Netherlands
2 Thermo Fisher Scientific, 5350 NE Dawson Creek Drive, Hillsboro, OR97124, USA
3 Thermo Fisher Scientific, Hannah-Kunath-StraBe 11, 28199 Bremen, Germany
Introduction: In order to understand protein function, it is critical to have access to a protein structure. Most of the currently available experimental structural information comes from either X-ray crystallography (XRD) or cryo-electron microscopy (Cryo-EM). However, neither of these techniques allow the user to directly correlate proteoform information such as post translational modifications to the protein conformation, which can be critical to fully understand protein function. The ability of mass spectrometry to provide unaveraged data allows for detection of multiple coexisting proteoforms or conformations. However, structural mass spectrometry techniques such as Ion Mobility or Hydrogen Deuterium Exchange MS provide inferred structure at best, and often still requires a detailed 3D protein structure in order to interpret the data.
Methods: Recently, low energy electron holography (LEEH) was shown to be able to image nanoscale particles (1). Critical to this breakthrough was the ability to prepare an ultraclean substrate, which was done through in vacuo in beam deposition using a mass spectrometer, after which the samples were transferred using a vacuum suitcase to a different experimental setup for imaging. Using a mass spectrometer to prepare samples also characterization and even selection of different species (proteoform, conformation or even one protein from a complex matrix) for subsequent imaging. Furthermore, since LEEH makes use of low energy electrons it is particularly well suited to image proteins, as there is little to no damage when these proteins are exposed to the electron beam.
Preliminary data: Here we present an integrated mass spec-based system that can image individual single particles with high contrast and over extended periods of time. Our system combines ion beam deposition of native proteins on a modified Q Exactive UHMR mass spectrometer with direct protein imaging capabilities using single particle LEEH within a single instrument. The instrument workflow consists of 3 consecutive steps: acquiring a native MS spectrum to select the protein or proteoform of interest, energy-controlled deposition of the mass selected protein on an ultraclean graphene monolayer at ultrahigh vacuum and image the deposited proteins on the substrate using LEEH. In order to validate the instrument and assess its performance we will show how our setup imaged various types of proteins over a broad size range. We will demonstrate how the resulting holograms can be reconstructed to generate 2D protein images and how due to the non-damaging nature of the low energy electrons, even videos can be acquired following the behavior and dynamics of the particles on the freestanding graphene. Finally, we show how single particle LEEH can image the structure and dynamics for a FAB-antigen complex (~70 kDa) that which was too small and dynamic for Cryo-EM studies, and where the conformational dynamics were absent in the crystal structure.
Novel aspect: Structural dynamics observed through low energy electron holography on proteins after ion beam deposition mass spectrometry on graphene supports.
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