Olguin-Arredondo Hector Armando, Vallejo-Cordoba Belinda, González-Córdova Aarón Fernando
Centro de Investigación en Alimentaciń y Desarrollo, A. C., Carretera a La Victoria, Hermosillo, Sonora, México.
J Capill Electrophor Microchip Technol. 2005;9(3-4):65-70.
The methodological aspects for the separation, fractionation, and peptide mapping by free zone capillary electrophoresis (CZE) of beta-lactoglobulin (beta-Lg) variants A and B were established. First, beta-Lg variants A or B were separated and fractionated by CZE. Then, the collected protein fraction was subjected to off-line tryptic digestion. Second, peptide mapping of the tryptic hydrolysates and peptide fraction collection were carried out by CZE. beta-Lg variants were separated and collected using an uncoated capillary (72 cm x 75 microm i.d.) in 0.05 M borate buffer containing 0.1% Tween 20 at pH 8.0 by applying 20 kV. By subjecting the capillary under pressure after a delay time of 15%, the protein was collected in a microvial containing digestion buffer. The most suitable conditions for the tryptic digestion of beta-Lg were established by monitoring the reaction products with CZE. A tryptic hydrolysis with an enzyme-to-substrate ratio (E/S) of 1/20 and incubation for 20 hr at 37 degrees C was found to result in the most suitable conditions. Peptides were separated and collected using an uncoated capillary (120 cm x 75 microm i.d.) in 0.15 M formic acid at pH 2.3 by applying 28 kV. Peptide maps were highly reproducible as shown by coefficients of variation of less than 0.89 and 5.42% for migration times and peak areas, respectively. Moreover, very good resolution of the peptide maps revealed the region in which the aberrant peptides of the beta-Lg variants may be located.
建立了通过自由区毛细管电泳（CZE）分离、分级和肽图谱分析β-乳球蛋白（β-Lg）变体A和B的方法学。首先，通过CZE分离和分级β-Lg变体A或B。然后，对收集的蛋白质级分进行离线胰蛋白酶消化。其次，通过CZE对胰蛋白酶水解产物进行肽图谱分析和肽级分收集。使用未涂层毛细管（72 cm×75μm内径），在pH 8.0的含0.1%吐温20的0.05 M硼酸盐缓冲液中，施加20 kV电压，分离并收集β-Lg变体。在延迟15%的时间后对毛细管施加压力，将蛋白质收集到含有消化缓冲液的微量瓶中。通过用CZE监测反应产物，确定了β-Lg胰蛋白酶消化的最适宜条件。发现酶与底物比（E/S）为1/20并在37℃孵育20小时的胰蛋白酶水解可得到最适宜条件。使用未涂层毛细管（120 cm×75μm内径），在pH 2.3的0.15 M甲酸中，施加28 kV电压，分离并收集肽段。肽图谱具有高度可重复性，迁移时间和峰面积的变异系数分别小于0.89%和5.42%。此外，肽图谱的良好分辨率揭示了β-Lg变体异常肽段可能所在的区域。
