Yang Pengxiang, Cooks R Graham, Ouyang Zheng, Hawkridge Adam M, Muddiman David C
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Anal Chem. 2005 Oct 1;77(19):6174-83. doi: 10.1021/ac050711l.
Gentle protein electrospray ionization is achieved using the high-velocity gas flow of an air amplifier to improve desolvation in conventional ESI and generate intact folded protein ions in the gas phase. Comparisons are made between the ESI spectra of a number of model proteins, including ubiquitin, cytochrome c, lysozyme, and myoglobin, over a range of pH values under optimized conditions, with and without using an air amplifier to achieve high-velocity gas flow. Previously reported increased ion signals are confirmed. In addition, the peaks recorded using the air amplifier are shown to be narrower, corresponding to more complete desolvation. Significant changes in the charge-state distribution also are observed, with a shift to lower charge state at high-velocity flow. The relationship between the observed charge-state distribution and protein conformation was explored by comparing the charge-state shifts and the distributions of charge states for proteins that are or are not stable in their native conformations in low pH solutions. The data suggest retention of native or nativelike protein conformations using the air amplifier in all cases examined. This is explained by a mechanism in which the air amplifier rapidly creates small droplets from the original large ESI droplets and these microdroplets then desolvate without a significant decrease in pH, resulting in retention of the folded protein conformations. Furthermore, the holoform of ionized myoglobin is visible at pH 3.5, a much lower value than the minimum needed to see this form in conventional ESI. These results provide evidence for the importance of the conditions used in the desolvation process for the preservation of the protein conformation and suggest that the conditions achieved when using high-velocity gas flows to assist droplet evaporation and ion desolvation are much gentler than those in conventional ESI experiments.
利用空气放大器的高速气流实现温和的蛋白质电喷雾电离，以改善传统电喷雾电离中的去溶剂化作用，并在气相中生成完整的折叠蛋白质离子。在优化条件下，在一系列pH值范围内，对包括泛素、细胞色素c、溶菌酶和肌红蛋白在内的多种模型蛋白质的电喷雾电离光谱进行了比较，分别采用和不采用空气放大器以实现高速气流的情况。先前报道的离子信号增加得到了证实。此外，使用空气放大器记录的峰更窄，这对应于更完全的去溶剂化。还观察到电荷态分布的显著变化，在高速气流下电荷态向较低电荷态转变。通过比较在低pH溶液中天然构象稳定或不稳定的蛋白质的电荷态变化和电荷态分布，探讨了观察到的电荷态分布与蛋白质构象之间的关系。数据表明，在所研究的所有情况下，使用空气放大器可保留天然或类似天然的蛋白质构象。这可以通过一种机制来解释，即空气放大器迅速将原始的大电喷雾电离液滴形成小液滴，然后这些微滴去溶剂化，而pH值没有显著降低，从而保留了折叠的蛋白质构象。此外，在pH 3.5时可以看到离子化肌红蛋白的全形，这一pH值远低于在传统电喷雾电离中看到这种形式所需的最低值。这些结果证明了去溶剂化过程中所使用的条件对保存蛋白质构象的重要性，并表明使用高速气流辅助液滴蒸发和离子去溶剂化时所实现的条件比传统电喷雾电离实验中的条件要温和得多。
