期刊
ANALYTICAL CHEMISTRY
卷 95, 期 48, 页码 17525-17532出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.3c02640
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This study demonstrates that 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) act as new ESI-compatible neutral pH buffers with buffering capacity at physiological pH. They can preserve noncovalent protein-ligand complexes in the gas phase and are suitable for studying multisubunit protein complexes.
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 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 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.
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