Effects of Ammonium Bicarbonate on the Electrospray Mass Spectra of Proteins: Evidence for Bubble-Induced Unfolding
Effects of Ammonium Bicarbonate on the Electrospray Mass Spectra of Proteins: Evidence for Bubble-Induced Unfolding
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DOI：
10.1021/ac401020s
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发表时间：
2013-07-02
影响因子：
7.4
通讯作者：
Konermann, Lars
中科院分区：
文献类型：
--
作者：
Hedges, Jason B.;Vahidi, Siavash;Konermann, Lars
通过电喷雾电离（ESI）质谱（MS）的许多蛋白质研究努力确保“天然”溶剂环境，即，非变性条件下的气相离子形成点。理想情况下，这些研究将采用挥发性pH缓冲液来减轻ESI期间可能发生的H+浓度变化。乙酸铵是一种常用的添加剂，尽管其在pH 7下的缓冲能力较低。碳酸氢铵大大提高了pH值的稳定性，从而提供了一种有趣的替代方案。令人惊讶的是，在pH 7的碳酸氢盐中的蛋白质分析往往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的蛋白质时获得的那些。这种效应以前已经报道过（Sterling，H. J.道：卡苏角一、Susa，A. C.的;威廉姆斯，E. R. Anal. 2012，84，3795），但其确切的机理起源仍不清楚。ESI介导的解折叠在其他相同条件下在乙酸盐中不发生。我们证明，加热含蛋白质的碳酸氢盐溶液的结果在广泛的泡沫，造成的CO2脱气。相反，乙酸盐溶液不产生泡沫。由气泡引起的蛋白质变性是众所周知的现象。吸附到气/液界面伴随着重大的构象变化，使蛋白质充当表面活性剂。啤酒的泡沫就是这种效应的表现。碰撞和黑体液滴加热促进了ESI过程中碳酸氢盐中气泡的形成。我们的数据表明，热和气泡协同作用，导致展开过程中的电喷雾，而蛋白质驻留在ESI液滴。由于这种效应，我们建议不要使用碳酸氢铵进行天然ESI-MS。乙酸铵代表了一种温和的液滴环境，尽管其缓冲能力较低。
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.