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.

天然电喷雾电离质谱（native electrospray ionization mass spectrometry, ESI-MS）已成为研究大分子复合物类天然结构的有力工具。尽管该技术应用广泛，但目前主流使用的“缓冲液”——乙酸铵（ammonium acetate, AmAc）——其共轭酸乙酸的解离常数（pKa）为4.75，氨的pKa为9.25，在生理pH范围（7.0~7.4）内几乎不具备缓冲能力。电喷雾诱导的氧化还原反应会改变电喷雾毛细管内液体的pH值，进而可能引发蛋白质解折叠或削弱pH敏感相互作用。因此，开发兼具挥发性、电喷雾兼容性，且能在生理pH范围内有效维持pH的缓冲液具有重要研究价值。
本研究证实，2,2-二氟乙胺（2,2-difluoroethylamine, DFEA）与2,2,2-三氟乙胺（2,2,2-trifluoroethylamine, TFEA）可在乙酸铵无法发挥缓冲作用的生理pH区间提供缓冲能力，二者共轭酸的pKa分别为7.2和5.5。针对模型蛋白质细胞色素c与肌红蛋白开展的天然电喷雾电离质谱实验表明，使用DFEA与TFEA进行电喷雾时，气相中非共价蛋白质-配体复合物得以完整保留。蛋白质稳定性测定与碰撞诱导解折叠实验进一步验证，DFEA与TFEA均不会在溶液或气相中降低模型蛋白质的稳定性。最后，本研究证明，借助天然电喷雾电离质谱技术，可在DFEA或TFEA存在的条件下研究多亚基蛋白质复合物，如乙醇脱氢酶与伴刀豆球蛋白A。本研究结果确立了DFEA与TFEA作为新型电喷雾兼容型中性pH缓冲液的地位，有望推动天然电喷雾电离质谱技术在大分子复合物分析中的应用，尤其是对pH波动敏感的复合物分析。