Chen Jinzhi, Lee Cheng S, Shen Yufeng, Smith Richard D, Baehrecke Eric H
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Electrophoresis. 2002 Sep;23(18):3143-8. doi: 10.1002/1522-2683(200209)23:18<3143::AID-ELPS3143>3.0.CO;2-7.
On-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed using a microinjector as the interface for performing two-dimensional (2-D) protein/peptide separations of complex protein mixtures. The focusing effect of CIEF not only contributes to a high-resolution protein/peptide separation, but also may permit the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. The preparative capabilities of CIEF are much larger than most of capillary-based electrokinetic separation techniques since the entire capillary is initially filled with a solution containing proteins/peptides and carrier ampholytes for the creation of a pH gradient inside the capillary. The focused peptides which have a similar pI are coinjected into the second separation dimension and further resolved by their differences in hydrophobicity. The resolving power of combined CIEF-CRPLC system is demonstrated using the soluble fraction of Drosophila salivary glands taken from a period beginning before steroid-triggered programmed cell death and extending to its completion. The separation mechanisms of CIEF and CRPLC are completely orthogonal and the overall peak capacity is estimated to be around approximately 1800 over a run time of less than 8 h. Significant enhancement in the separation peak capacity can be realized by further increasing the number of CIEF fractions and/or slowing the solvent gradient in CRPLC, however, at the expense of overall analysis time. The results of our preliminary studies display significant differences in the separation profiles of peptide samples obtained from salivary glands of animals staged at the 6 and 12 h following puparium formation.
利用微注射器作为接口，开发了毛细管等电聚焦（CIEF）与毛细管反相液相色谱（CRPLC）的在线联用方法，用于对复杂蛋白质混合物进行二维（2-D）蛋白质/肽分离。CIEF的聚焦作用不仅有助于实现高分辨率的蛋白质/肽分离，还可能允许分析低丰度蛋白质，其典型的浓缩系数为50-100倍。CIEF的制备能力比大多数基于毛细管的电动分离技术大得多，因为整个毛细管最初充满了含有蛋白质/肽和载体两性电解质的溶液，用于在毛细管内创建pH梯度。具有相似pI的聚焦肽被共注入到第二分离维度，并根据它们在疏水性上的差异进一步分离。使用从类固醇触发的程序性细胞死亡开始前到结束的时间段内采集的果蝇唾液腺可溶部分，证明了CIEF-CRPLC联用系统的分离能力。CIEF和CRPLC的分离机制完全正交，在不到8小时的运行时间内，总峰容量估计约为1800。通过进一步增加CIEF馏分的数量和/或减缓CRPLC中的溶剂梯度，可以实现分离峰容量的显著提高，然而，这是以牺牲整体分析时间为代价的。我们初步研究的结果显示，从蛹形成后6小时和12小时的动物唾液腺中获得的肽样品的分离图谱存在显著差异。
