Femtomolar concentration detection limit and zeptomole mass detection limit for protein separation by capillary isoelectric focusing and laser-induced fluorescence detection.
Femtomolar concentration detection limit and zeptomole mass detection limit for protein separation by capillary isoelectric focusing and laser-induced fluorescence detection.
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DOI：
10.1021/ac8025948
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发表时间：
2009-03-01
影响因子：
7.4
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Ramsay, Lauren M.;Dickerson, Jane A.;Dada, Oluwatosin;Dovichi, Norman J.
对于大多数形式的毛细管电泳而言，荧光往往产生最低的检测下限。有两个问题阻碍了它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，将阳离子残基转化为中性或阴离子产物。充其量，这些试剂会扰乱蛋白质的等电点。更糟糕的是，他们将每种蛋白质转化为数百种不同的荧光产物，这扰乱了分析。第二个问题是商业两性离子中的杂质产生的大背景信号。这种背景信号在光谱的蓝色部分被激发时特别强，这是许多常见的荧光标记试剂所需要的。本文对这些问题进行了研究。对于标签，我们使用Chromeo P540，这是一种将阳离子赖氨酸残基转化为阳离子荧光产品的荧光试剂。反应产物在绿色中被激发，这降低了两性阳离子中存在的杂质产生的背景信号。为了进一步降低背景信号，我们用大功率光电二极管对两性染料进行了光漂白。光漂白将两性离子空白中的噪音降低了一个数量级。用pH为3~10的光漂白两性离子等电聚焦，对Chromeo P540标记的β-乳球蛋白进行等电聚焦，其浓度检出限为270±25fM，质量检出限为150±15zmol。浓度检出限为520±40fM，质量检出限为310±30zmol，pH为4~8。从Barrett‘s食道细胞系制备匀浆，用毛细管等电聚焦分离，可重复性地产生数十个峰。用于分离的样品与来自三个细胞的标记蛋白匀浆相同。
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 β-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.
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影响因子：
7.4
作者：
Hu, S;Michels, DA;Dovichi, NJ
通讯作者：
Dovichi, NJ
影响因子：
14.9
作者：
CHEN, DY;HARKE, HR;DOVICHI, NJ
通讯作者：
DOVICHI, NJ
影响因子：
7.4
作者：
Liu, Z;Pawliszyn, J
通讯作者：
Pawliszyn, J
DOI：
10.1016/s0021-9673(01)94519-4
发表时间：
1987-01-30
期刊：
JOURNAL OF CHROMATOGRAPHY
影响因子：
--
作者：
HJERTEN, S;LIAO, JL;YAO, K
通讯作者：
YAO, K
DOI：
10.1016/0165-022x(82)90013-6
发表时间：
1982-01-01
影响因子：
--
作者：
BJELLQVIST, B;EK, K;POSTEL, W
通讯作者：
POSTEL, W