Liu Hechun, Yang Chun, Yang Qing, Zhang Weibing, Zhang Yukui
National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Mar 5;817(1):119-26. doi: 10.1016/j.jchromb.2004.04.038.
A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.
报道了一种新型的蛋白质二维（2D）分离系统。在该系统中，采用一段透析中空纤维膜作为毛细管等电聚焦（CIEF）和毛细管非凝胶筛分电泳（CNGSE）在线联用的接口。该系统通过将二维聚丙烯酰胺凝胶电泳（2D PAGE）的分离原理转移到毛细管形式，与二维聚丙烯酰胺凝胶电泳等效。蛋白质在第一维CIEF中根据其等电点（pI）的差异进行聚焦和分离。聚焦的蛋白质区带转移到透析中空纤维接口，在那里蛋白质与十二烷基硫酸钠（SDS）进行疏水复合。带负电荷的蛋白质进行电迁移，并在第二维CNGSE中根据其大小差异进一步分离，其中使用葡聚糖溶液（一种可替代的筛分基质，而非交联聚丙烯酰胺凝胶）进行基于大小的蛋白质分离。这两种技术的联用归因于透析膜接口的高效性。通过分别对大鼠肺癌细胞分泌的血红蛋白和蛋白质混合物进行分离来检验每种技术，证明了CIEF-CNGSE联用分离技术的可行性和正交性，这是使峰容量或分辨率元素最大化的一个重要因素。发现二维分离策略比单独一维获得的分辨率和总峰容量大大提高。
