Thermal unfolding of proteins probed by laser spray mass spectrometry.
Thermal unfolding of proteins probed by laser spray mass spectrometry.
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DOI：
10.1002/rcm.3517
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发表时间：
2008-05
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影响因子：
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通讯作者：
Xiangguo Shi;Atsushi Takamizawa;Y. Nishimura;K. Hiraoka;S. Akashi
中科院分区：
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作者：
Xiangguo Shi;Atsushi Takamizawa;Y. Nishimura;K. Hiraoka;S. Akashi
迄今为止，已经通过各种分析方法对细胞色素c (cyt c) 在不同温度和pH 下的稳定性和构象变化进行了充分研究。我们发现，与其他方法相比，激光喷雾质谱法能够更快、更方便地监测 cyt c 的变化。结果与相对酸性条件下的圆二色性（CD）实验很好地相关，而相对酸性条件使蛋白质不稳定。在不同激光功率水平下获得了不同 pH 值下细胞色素 c 的激光喷雾质谱。在激光喷雾质谱中观察到蛋白质的双峰电荷态分布，表明结构变化的双态模型；较低的电荷对应于折叠状态，较高的电荷对应于展开状态。基于该结果，将蛋白质的假定变性曲线绘制为激光功率的函数，并且在每个pH下获得假定50%的蛋白质变性的激光功率（E50%）。我们还使用 CD 光谱检查了不同 pH 值下细胞色素 c 的解链温度 Tm。 E50%与细胞色素c的Tm之间的相关系数为0.999，显示出良好的相关性。此外，对泛素进行激光喷雾分析发现其比 cyt c 更热稳定，其 E50% 高于 cyt c。这些结果表明，激光喷雾质谱法可以成为一种极其方便的方法，用于探测由于 pH 的轻微变化而引起的细微结构差异的蛋白质的热稳定性和动态构象变化。
The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.