Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
Anal Chem. 2013 Jul 2;85(13):6469-76. doi: 10.1021/ac401020s. Epub 2013 Jun 18.
Many protein investigations by electrospray ionization (ESI) mass spectrometry (MS) strive to ensure a "native" solvent environment, i.e., nondenaturing conditions up to the point of gas-phase ion formation. Ideally, these studies would employ a volatile pH buffer to mitigate changes in H(+) concentration that can occur during ESI. Ammonium acetate is a commonly used additive, despite its low buffering capacity at pH 7. Ammonium bicarbonate provides greatly improved pH stabilization, thus offering an interesting alternative. Surprisingly, protein analyses in bicarbonate at pH 7 tend to result in the formation of very high charge states, similar to those obtained when electrospraying unfolded proteins in a denaturing solvent. This effect has been reported previously (Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795), but its exact mechanistic origin remains unclear. ESI-mediated unfolding does not take place in acetate under otherwise identical conditions. We demonstrate that heating of protein-containing bicarbonate solutions results in extensive foaming, caused by CO2 outgassing. In contrast, acetate solutions do not generate foam. Protein denaturation caused by gas bubbles is a well-known phenomenon. Adsorption to the gas/liquid interface is accompanied by major conformational changes that allow the protein to act as a surfactant. The foaming of beer is a manifestation of this effect. Bubble formation in bicarbonate during ESI is facilitated by collisional and blackbody droplet heating. Our data imply that heat and bubbles act synergistically to cause unfolding during the electrospray process, while proteins reside in ESI droplets. Because of this effect we advise against the use of ammonium bicarbonate for native ESI-MS. Ammonium acetate represents a gentler droplet environment, despite its low buffering capacity.
许多通过电喷雾电离 (ESI) 质谱 (MS) 进行的蛋白质研究都致力于确保“天然”溶剂环境，即在达到气相离子形成之前保持非变性条件。理想情况下，这些研究将采用挥发性 pH 缓冲剂来减轻 ESI 过程中可能发生的 H+浓度变化。尽管在 pH 7 时乙酸铵的缓冲能力较低，但它仍是一种常用的添加剂。碳酸氢铵提供了极大的 pH 稳定性改进，因此是一种有趣的替代选择。令人惊讶的是，在 pH 7 的碳酸氢盐中进行蛋白质分析往往会导致形成非常高的电荷状态，类似于在变性溶剂中电喷雾未折叠蛋白质时获得的电荷状态。先前已经报道了这种效应（Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795），但其确切的机制起源仍不清楚。在其他条件相同的情况下，乙酸盐中不会发生 ESI 介导的展开。我们证明，含有碳酸氢盐的蛋白质溶液加热会导致 CO2 逸出引起大量泡沫。相比之下，乙酸盐溶液不会产生泡沫。由气泡引起的蛋白质变性是一种众所周知的现象。气泡吸附到气/液界面伴随着使蛋白质充当表面活性剂的主要构象变化。啤酒的泡沫就是这种效果的表现。在 ESI 过程中，碳酸氢盐中的气泡形成是由于碰撞和黑体液滴加热而促进的。我们的数据表明，在电喷雾过程中，热和气泡协同作用导致蛋白质在 ESI 液滴中展开。由于这种效应，我们建议避免在原生 ESI-MS 中使用碳酸氢铵。尽管乙酸铵的缓冲能力较低，但它代表了一种更温和的液滴环境。
