Shen Y, Berger S J, Anderson G A, Smith R D
Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Anal Chem. 2000 May 1;72(9):2154-9. doi: 10.1021/ac991367t.
Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ microL were detectable using conventional UV detection. CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels. Very small differences in peptide isoelectric points (deltapI approximately 0.01) could be resolved, High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.
目前正在开发几种用于全球蛋白质组表征的方法，这些方法基于对（通常是复杂的）蛋白质混合物消化产生的多肽混合物的分析。需要改进的肽分析方法，以实现样品浓缩、更高分辨率的分离，并与质谱直接兼容。在这项工作中，已经开发出了用于肽的高效毛细管等电聚焦（CIEF）分离的方法，该方法可根据肽的等电点同时进行样品浓缩和分离。在典型的非变性CIEF条件下，肽可浓缩约500倍，使用传统紫外检测可检测到浓度低于1 ng/μL的肽。与传统的凝胶等电聚焦相比，肽的CIEF分离提供了更快的等电点测量。肽等电点的非常小的差异（ΔpI约为0.01）都可以分辨出来。从酵母细胞质组分的胰蛋白酶消化中获得了复杂肽混合物的高效CIEF分离，并且与使用毛细管区带电泳和填充毛细管反相液相色谱获得的分离相比有显著改进。
