Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Anal Chem. 2009 Mar 1;81(5):1741-6. doi: 10.1021/ac8025948.
Fluorescence tends to produce the lowest detection limits for most forms of capillary electrophoresis. Two issues have discouraged its use in capillary isoelectric focusing. The first issue is fluorescent labeling of proteins. Most labeling reagents react with lysine residues and convert the cationic residue to a neutral or anionic product. At best, these reagents perturb the isoelectric point of the protein. At worse, they convert each protein into hundreds of different fluorescent products that confound analysis. The second issue is the large background signal generated by impurities within commercial ampholytes. This background signal is particularly strong when excited in the blue portion of the spectrum, which is required by many common fluorescent labeling reagents. This paper addresses these issues. For labeling, we employ Chromeo P540, which is a fluorogenic reagent that converts cationic lysine residues to cationic fluorescent products. The reaction products are excited in the green, which reduces the background signal generated by impurities present within the ampholytes. To further reduce the background signal, we photobleach ampholytes with high-power photodiodes. Photobleaching reduced the noise in the ampholyte blank by an order of magnitude. Isoelectric focusing performed with photobleached pH 3-10 ampholytes produced concentration detection limits of 270 +/- 25 fM and mass detection limits of 150 +/- 15 zmol for Chromeo P540 labeled beta-lactoglobulin. Concentration detection limits were 520 +/- 40 fM and mass detection limits were 310 +/- 30 zmol with pH 4-8 ampholytes. A homogenate was prepared from a Barrett's esophagus cell line and separated by capillary isoelectric focusing, reproducibly generating dozens of peaks. The sample taken for the separation was equal to the labeled protein homogenate from three cells.
荧光倾向于产生大多数形式的毛细管电泳的最低检测限。有两个问题阻碍了其在毛细管等电聚焦中的应用。第一个问题是蛋白质的荧光标记。大多数标记试剂与赖氨酸残基反应，并将阳离子残基转化为中性或阴离子产物。在最好的情况下，这些试剂会扰乱蛋白质的等电点。在最坏的情况下，它们将每个蛋白质转化为数百种不同的荧光产物，使分析变得复杂。第二个问题是商业两性电解质中的杂质产生的大背景信号。当用许多常见的荧光标记试剂所需的光谱的蓝色部分激发时，这种背景信号特别强。本文解决了这些问题。对于标记，我们使用 Chromeo P540，它是一种荧光试剂，将阳离子赖氨酸残基转化为阳离子荧光产物。反应产物在绿色中被激发，这降低了两性电解质中杂质产生的背景信号。为了进一步降低背景信号，我们用高功率光电二极管对两性电解质进行光漂白。光漂白将两性电解质空白中的噪声降低了一个数量级。用光漂白的 pH 3-10 两性电解质进行等电聚焦，可产生 270±25 fM 的浓度检测限和 150±15 zmol 的质量检测限，用于标记β-乳球蛋白的 Chromeo P540。用 pH 4-8 的两性电解质，浓度检测限为 520±40 fM，质量检测限为 310±30 zmol。从 Barrett 食管细胞系制备匀浆液，并通过毛细管等电聚焦分离，可重复地产生数十个峰。用于分离的样品与来自三个细胞的标记蛋白匀浆液相同。
