3D Structure Determination of Protein Complexes using Matrix-Landing Mass Spectrometry

Native mass spectrometry (MS) is an emerging technology that can provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe use of a chemical landing matrix that both preserves and protects the structural integrity of the deposited particles. With this system we obtained the first 3D reconstructed structure of gas-phase, deposited biomolecular ions – the 800 KDa protein complex GroEL. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.

Fluorescence-based Techniques to Study the Structure and Dynamics of Mass-selected Biomolecular Ions

Laser-induced fluorescence studies on mass-selected biomolecules are a promising route to understand their properties in the gas phase and probe their intrinsic properties in a solvent-free environment. Fluorescence has been used to investigate the conformation and dynamics of gaseous biomolecular ions. With Förster Resonance Energy Transfer (FRET), it is now possible to obtain sensitive intramolecular distance information from large biomolecules, like proteins, with high chemical specificity. With growing interest and applications, gas-phase fluorescence measurements can shed greater light on the characteristics of proteins in the gas phase. Compared to the solution phase measurements, gas-phase fluorescence can also help understand the influence of solvent interactions on the protein structure and function.

Combining ultra-high resolution ion mobility spectrometry with cryogenic IR spectroscopy for the study of biomolecular ions

We explore the capability of SLIM-based IMS for isomer selectivity in combination with cryogenic, messenger-tagging IR spectroscopy.