Fluorinated Ethylamines as Electrospray-Compatible Neutral pH Buffers for Native Mass Spectrometry

Abstract

Native electrospray ionization mass spectrometry (ESI-MS) has emerged as a potent tool for examining the native-like structures of macromolecular complexes. Despite its utility, the predominant "buffer" used, ammonium acetate (AmAc) with pK a values of 4.75 for acetic acid and 9.25 for ammonium, provides very little buffering capacity within the physiological pH range of 7.0−7.4. ESI-induced redox reactions alter the pH of the liquid within the ESI capillary. This can result in protein unfolding or weakening of pHsensitive interactions. Consequently, the discovery of volatile, ESIcompatible buffers, capable of effectively maintaining pH within a physiological range, is of high importance. Here, we demonstrate that 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) offer buffering capacity at physiological pH where AmAc falls short, with pK a values of 7.2 and 5.5 for the conjugate acids of DFEA and TFEA, respectively. Native ESI-MS experiments on model proteins cytochrome c and myoglobin electrosprayed with DFEA and TFEA demonstrated the preservation of noncovalent protein−ligand complexes in the gas phase. Protein stability assays and collision-induced unfolding experiments further showed that neither DFEA nor TFEA destabilized model proteins in solution or in the gas phase. Finally, we demonstrate that multisubunit protein complexes such as alcohol dehydrogenase and concanavalin A can be studied in the presence of DFEA or TFEA using native ESI-MS. Our findings establish DFEA and TFEA as new ESI-compatible neutral pH buffers that promise to bolster the use of native ESI-MS for the analysis of macromolecular complexes, particularly those sensitive to pH fluctuations.

Differentiating specific and non-specific protein-metabolite interactions using gradient open port probe electrospray ionization mass spectrometry

“…With the simplicity of sample preparation, low sample consumption, high sensitivity, and high throughput, native ESI-MS has received a lot of attention in drug screening. 1,2,24,25 To optimize the native ESI-MS to explore PMIs, a variety of studies have been reported, including the type of ion source, 21,26,27 the size of ESI tips, [28][29][30][31] the buffer maintaining the native conditions, [32][33][34] and solvent additives. 22,35,36 However, only a few studies have dealt with the issue of non-specific binding between the target protein and the small molecules tested.…”

MD Simulations of Peptide-Containing Electrospray Droplets: Effects of Parameter Settings on the Predicted Mechanisms of Gas Phase Ion Formation

“…The final steps of the ESI process, i.e., the release of gaseous analyte ions from highly charged nanodroplets, is a topic of great interest and considerable controversy. − Three mechanisms have emerged as likely ion formation pathways . In the charged residue mechanism (CRM), analyte ions are released upon droplet evaporation to dryness. , The ion evaporation mechanism (IEM) proceeds with analyte ejection from the droplet, driven by Coulombic repulsion between the analyte charge and other charges on the droplet. , The chain ejection mechanism (CEM) applies to disordered polymeric analytes (such as unfolded proteins), which gradually extrude from the droplet and end up as highly charged and electrostatically stretched gaseous ions. ,, In addition to these mechanisms, various hybrid scenarios may exist. , …”