Chen Jinzhi, Gao Jun, Lee Cheng S
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
J Proteome Res. 2003 May-Jun;2(3):249-54. doi: 10.1021/pr0255811.
Capillary isoelectric focusing (CIEF) involves the use of the entire capillary filled with a mixture containing protein/peptide analytes and carrier ampholytes. Thus, the preparative capabilities of CIEF are inherently greater than most capillary-based electrokinetic separation techniques. To further increase sample loading and, therefore, the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from a solution reservoir, is demonstrated using a low p/ protein calibration kit and tryptic peptides from Saccharomyces cerevisiae. The proteins/peptides continuously migrate into the capillary and encounter a pH gradient established by carrier ampholytes originally present in the capillary for focusing and separation. Dynamic introduction and focusing in CIEF can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Depending on the mobilities of yeast peptides, the loading capacity of each peptide is measured to be around 8 to 45-fold of that obtained in conventional CIEF. By comparing with the concentrations of dilute yeast peptides originally present in the reservoir, an overall concentration factor of 1400-7700 together with excellent separation resolution is achieved using dynamic introduction and focusing. This concentration effect is further illustrated by detecting 10 pg/microL of bradykinin peptide spiked in yeast protein digest using only ultraviolet absorption.
毛细管等电聚焦（CIEF）涉及使用充满含有蛋白质/肽分析物和载体两性电解质混合物的整个毛细管。因此，CIEF的制备能力本质上大于大多数基于毛细管的电动分离技术。为了进一步增加样品加载量，从而提高聚焦分析物的浓度，使用低pI蛋白质校准试剂盒和来自酿酒酵母的胰蛋白酶肽，展示了一种基于从溶液储液器电动注入蛋白质/肽的动态方法。蛋白质/肽持续迁移到毛细管中，并遇到由最初存在于毛细管中的载体两性电解质建立的pH梯度，用于聚焦和分离。CIEF中的动态引入和聚焦可以通过各种电动条件直接控制，包括注入时间和施加的电场强度。样品加载量的差异由电动注入偏差造成，并受单个分析物的电泳迁移率影响。根据酵母肽的迁移率，测得每种肽的加载能力约为传统CIEF中获得的加载能力的8至45倍。通过与储液器中最初存在的稀释酵母肽的浓度进行比较，使用动态引入和聚焦实现了1400 - 7700的总体浓缩因子以及出色的分离分辨率。通过仅使用紫外吸收检测添加到酵母蛋白消化物中的10 pg/μL缓激肽肽，进一步说明了这种浓缩效应。
