The effects of solution additives and gas‐phase modifiers on the molecular environment and conformational space of common heme proteins

The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas-phase modifiers on biomolecules characterized using ion mobility techniques.The effect of solution additives and gas-phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100-500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time-of-flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization.Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas-phase modifiers (i.e., N2 doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas-modifier mass and size and changes in the relative abundances and number of IMS bands are observed.We attribute the observed changes in the mobility profiles in the presence of gas-phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.