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

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 pKa 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
pH-sensitive interactions. Consequently, the discovery of volatile,
ESI-compatible 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 pKa 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.