Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry

Abstract

Noncovalent interactions between biomolecules are critical to their activity. Native mass spectrometry (MS) has enabled characterization of these interactions by preserving noncovalent assemblies for mass analysis, including protein–ligand and protein–protein complexes for a wide range of soluble and membrane proteins. Recent advances in native MS of lipoprotein nanodiscs have also allowed characterization of antimicrobial peptides and membrane proteins embedded in intact lipid bilayers. However, conventional native electrospray ionization (ESI) can disrupt labile interactions. To stabilize macromolecular complexes for native MS, charge reducing reagents can be added to the solution prior to ESI, such as triethylamine, trimethylamine oxide, and imidazole. Lowering the charge acquired during ESI reduces Coulombic repulsion that leads to dissociation, and charge reduction reagents may also lower the internal energy of the ions through evaporative cooling. Here, we tested a range of imidazole derivatives to discover improved charge reducing reagents and to determine how their chemical properties influence charge reduction efficacy. We measured their effects on a soluble protein complex, a membrane protein complex in detergent, and lipoprotein nanodiscs with and without embedded peptides, and used computational chemistry to understand the observed charge-reduction behavior. Together, our data revealed that hydrophobic substituents at the 2 position on imidazole can significantly improve both charge reduction and gas-phase stability over existing reagents. These new imidazole derivatives will be immediately beneficial for a range of native MS applications and provide chemical principles to guide development of novel charge reducing reagents.

Cryo-EM structure of arabinosyltransferase EmbB from Mycobacterium smegmatis

“…EmbB was buffer exchanged into 0.2 M ammonium acetate at pH 6.8 (Sigma-Aldrich) with either 0.01% C12E8 (Anatrace) or 0.02% DDM (Anatrace) detergent using gel filtration. Native mass spectrometry (MS) analysis of EmbB in C12E8 detergent was performed using a Q-Exactive HF Orbitrap with Ultra High Mass Range modifications (Thermo Fisher Scientific) using previously described methods 70 . The HCD voltage was set to 150 V, and the capillary temperature was increased to 300°C.…”

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

“…To avoid this preferential dissociation, we tested imidazole derivatives that have previously been shown to stabilize nanodisc complexes. 35 We found that 2-methylimidazole stabilized POPC/SOPE nanodiscs the most and caused POPC/SOPE nanodiscs to have higher mass distributions, indicating less dissociation (Figure 1). Interestingly, the average lipid mass shows that 2methylimidazole retained the labile SOPE up to 200 V applied in the HCD cell ( Figure S1).…”

Influenza A M2 Channel Oligomerization is Sensitive to its Chemical Environment

“…The native mass spectra were deconvolved and quantified using UniDec, and macromolecular mass defect analysis was used to quantify the stoichiometries of AM2 and amantadine in nanodiscs. 39,50,51 Full details are provided in the Supporting Information.…”

“…The native mass spectra were deconvolved and quantified using UniDec, and macromolecular mass defect analysis was used to quantify the stoichiometries of AM2 and amantadine in nanodiscs. 41,61,62 Full details are provided in the Supporting Information. Prior published results with streptavidin, a similarly sized tetramer, with similar instrument conditions provided a positive control demonstrating the ability of native MS to preserve and detect specific noncovalent complexes of the same size.…”