毛细管等电聚焦电泳(cIEF)及其理论模拟计算新进展

Progress in capillary isoelectric focusing (cIEF) and theoretical simulation of its process

等电聚焦电泳(IEF)是一种在一个pH梯度内操作的特殊电泳。IEF是表征蛋白质电荷不均一性的强有力工具。在一个pH梯度内,酸碱两性物(例如蛋白质)在电场的作用下会被分离,并最后在pH梯度内pH值与两性物等电点(pI)值相等的地方被聚焦成很窄的区段。IEF的分辨率在所有基于电荷的分离技术中是最高的。薄层凝胶IEF从它在上世纪70年代初被发明的时间起就成为在生物实验室被广泛应用的分析技术。但薄层凝胶IEF的主要问题是不能做定量分析,全靠手工操作及分析速度低。当毛细管IEF(cIEF)在1985年被报道时,科学家们就都立即认为cIEF能够克服IEF所有的问题。虽然cIEF看起来有很多优势,但是20多年后的今天它并没有被生物实验室广泛接受为薄层凝胶IEF的替代技术。究其原因是使用通用毛细电泳仪器的cIEF在操作上有很多困难。这种技术在IEF过程后还需要一个额外的移动过程来把聚在毛细管分离柱内的蛋白质区段推到检测器的检测点。这是由于这些毛细电泳仪器都是使用单点检测器。检测点都是靠近毛细管的一端。这个移动过程破坏IEF的高分辨率,降低重现性并使得分析速度变慢,造成在方法的优化上耗很多时间。全柱检测cIEF是在1992年提出的。该技术结合了凝胶IEF及毛细管分离的全部优点;如定量和自动化。它的分离速度大大提高。本文简单综述cIEF对不同生物实验室样品的应用。这些样品包括单克隆抗体,重组蛋白质,蛋白质复合物及失活病毒。最后,本文简述cIEF理论模拟计算的新进展。

Iseelectric focusing （IEF） was seen as a special electrophoresis technology performed in a pH gradient that could be a pewerful tool for profiling protein charge heterogeneity. In the pH gradient, ampholytes such as proteins are separated and focused into narrow bands at their isoelectric points （pIs） under an electrical field. IEF had superior resolution compared to other charge based separation technologies for proteins. Slab gel IEF became a popular technique in biological laboratories since early1970＇s. However, there are limitations regarding its lack of quantitation, automation and speed. It appeared that these would be overcome by IEF performed in capillary format （cIEF） that was first reported in 1985. Despite cIEF＇s inherent appeal, its widespread acceptance to replace slab gel IEF in biological laboratories has not occurred after more than 20 years, due to performance and procedural difficulties when cIEF is performed on capillary electrophoresis （CE） instruments （conventional clEF）. These instruments use a single point detection system located on one end of the capillary column. After the IEF process is complete, a mobilization phase is required to move the focused protein zones past the single point detector. This can result in poor separation resolution, poor reproducibility and significantly longer time to develop methods and analyze samples. Whole - column detection cIEF was introduced in 1992. This technology combined the benefits of gel like IEF separation and detection with the automation and quantitation of column based separation, but at a significantly faster speed. The applications of cIEF in biotechnology laboratories, including analysis of monoclonal antibodies, recombinant proteins, protein conjugates and viruses, are reviewed in the paper. Finally, progress in theories and cIEF process simulation is introduced.