Laboratoire de Physicochimie des Electrolytes, Colloïdes et Sciences Analytiques, UMR CNRS-UPMC, Ecole Nationale Supérieure de Chimie de Paris, 75231 Paris, France.
Electrophoresis. 2009 Dec;30(23):4040-8. doi: 10.1002/elps.200900091.
A new online coupling of CIEF with ESI-MS has been developed in glycerol-water media. This improved protocol provides: (i) the electric continuity during the whole analysis by a discontinuous filling of the capillary with 60:40 (cm/cm) catholyte/proteins-ampholyte mixture; (ii) the use of an anticonvective medium, i.e. 30:70 glycerol/water, v/v, compatible with MS detection and as an aid to hydrophobic protein solubilization and (iii) the use of unmodified bare fused-silica capillaries, as the glycerol/water medium strongly reduces EOF. Focusing was performed in positive polarity and cathodic mobilization was achieved by both voltage and pressure application. The setup was optimized with respect to analysis time, sensitivity and precision on pI determination. The optimized anolyte and catholyte were composed of 50 mM formic acid/1 mM glutamic acid (pH 2.35) and 100 mM NH(3)/1 mM lysine (pH 10.6), respectively. The effects of ampholyte concentration, focusing time and ESI parameters were presented for model proteins and discussed. This new integrated protocol should be an easy and effective additional tool in the field of proteome analysis, providing a means for the characterization of a large number of hydrophilic and hydrophobic proteins.
已在甘油-水介质中开发出一种新的毛细管等电聚焦（CIEF）与电喷雾质谱（ESI-MS）在线联用方法。该改进方案具有以下优点：（i）通过用 60:40（cm/cm）阴极液/蛋白质-两性电解质混合物间歇填充毛细管，实现整个分析过程中的电流连续性；（ii）使用非对流介质，即 30:70 甘油/水（v/v），与 MS 检测兼容，并有助于疏水性蛋白质的溶解；（iii）使用未经修饰的裸熔融石英毛细管，因为甘油/水介质强烈降低了电渗流。聚焦在正极性下进行，通过施加电压和压力实现阴极迁移。根据 pI 测定的分析时间、灵敏度和精密度对该装置进行了优化。优化的阳极液和阴极液分别由 50 mM 甲酸/1 mM 谷氨酸（pH 2.35）和 100 mM NH₃/1 mM 赖氨酸（pH 10.6）组成。还针对模型蛋白质介绍并讨论了两性电解质浓度、聚焦时间和 ESI 参数的影响。该新的集成方案应成为蛋白质组分析领域中一种简单有效的附加工具，为大量亲水和疏水蛋白质的特性描述提供了一种手段。
