Denaturation of lysozyme and myoglobin in laser spray
Denaturation of lysozyme and myoglobin in laser spray
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DOI：
10.1016/j.jasms.2005.02.012
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发表时间：
2005-06
影响因子：
3.2
通讯作者：
Atsushi Takamizawa;S. Fujimaki;J. Sunner;K. Hiraoka
中科院分区：
文献类型：
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作者：
Atsushi Takamizawa;S. Fujimaki;J. Sunner;K. Hiraoka
在激光喷雾中，电喷雾毛细管的尖端被连续的CO2激光束照射。在这里，我们报告的结果，从一个修改后的激光喷涂方法，采用相对较低的激光辐照度水平。当激光功率为10.2W，焦斑尺寸为10.3mm，覆盖整个毛细管前表面时，辐照度为10.3 × 103 W/cm ~ 2。这导致水溶液的静止和平稳蒸发。这种“蒸发模式”激光喷雾方法产生了迄今为止在我们实验室中使用激光照射电喷雾获得的最佳结果，产生更高和更稳定的信号。方法用于溶菌酶和肌红蛋白水溶液的分析。获得质谱作为激光功率从0 W（电喷雾）到102 W的函数。用CCD相机观察在不锈钢毛细管尖端处产生的喷雾。随着激光功率的增加，喷雾中的液滴变得更细，泰勒锥逐渐变小。当样品溶液仅从毛细管尖端略微突出时，记录到最强的离子信号。用2 W的激光功率观察到溶菌酶电荷态分布的增宽，这归因于蛋白质解折叠。当激光功率达到1.6W时，肌红蛋白不发生变性。然而，在1.8 W时观察到变性的突然发生，这是由于肌红蛋白电荷分布的加宽和脱辅基肌红蛋白峰的出现。这些发现表明，激光喷雾能够选择性地解离非共价复合物而不破坏共价键。
In laser spray, the tip of an electrospray capillary is irradiated with a continuous CO2laser beam. Here, we report results from a modified laser spray method that employs a relatively low laser irradiance level. With a laser power of ∼2 W and a focal spot size (∼0.3 mm), which covered the entire front surface of the electrospray capillary, the irradiance was ∼3 × 103W/cm2. This resulted in a quiescent and smooth vaporization of aqueous solutions. This “evaporation-mode” laser spray method yielded the best results so far obtained in our laboratory with laser-irradiated electrospray, producing higher and more stable signals. The method was applied to the analysis of aqueous solutions of lysozyme and myoglobin. Mass spectra were obtained as a function of laser power from 0 W (electrospray) to ∼2 W. The spray generated at the tip of the stainless steel capillary was observed with a CCD camera. With increase of laser power, the droplets in the spray became finer and the Taylor cone became progressively smaller. The strongest ion signals were recorded when the sample solution protruded only slightly from the tip of the capillary. A broadening of the lysozyme charge-state distribution, attributable to protein unfolding, was observed with a laser power of 2 W. No denaturation of myoglobin took place up to a laser power of 1.6 W. However, a sudden onset of denaturation was observed at 1.8 W as a broadening of the myoglobin charge distribution and the appearance of apo-myoglobin peaks. These findings demonstrate that laser spray is capable of dissociating the noncovalent complexes selectively without breaking covalent bonds.