Isobe T, Uchida K, Taoka M, Shinkai F, Manabe T, Okuyama T
Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Japan.
J Chromatogr. 1991 Dec 27;588(1-2):115-23. doi: 10.1016/0021-9673(91)85013-6.
An automated two-dimensional liquid chromatographic system was developed for systematic protein separations which could serve for analytical mapping and preparative separations of proteins. The system applies the principles of the column-switching technique, and consists of two different columns connected in tandem through an electrical column switching valve, two pumping systems to operate each column independently and a system controller to perform sequential chromatography on the two columns. A protein mixture is applied to the first-dimensional anion-exchange column and is separated by stepwise elution with an increasing sodium chloride concentration. The eluent is introduced directly to the second-dimensional reversed-phase column, and is further separated by gradient elution with an increasing acetonitrile concentration. The two elution stages are synchronized by a computer program. By this system, very complex protein mixtures such as crude cerebellar extracts were resolved reproducibly into ca. 200 peaks within 12 h. The method can be used for the total analysis of proteins in various tissues and cells without complicated premanupulation of samples, and allows the simultaneous analysis of a protein isolated by chromatography. The isolated protein is most suitable for use in the strategy of protein and gene sequence analysis.
开发了一种自动化二维液相色谱系统用于系统的蛋白质分离，该系统可用于蛋白质的分析图谱绘制和制备分离。该系统应用了柱切换技术原理，由通过电动柱切换阀串联连接的两根不同色谱柱、两个独立操作各色谱柱的泵系统以及一个在两根色谱柱上进行顺序色谱分析的系统控制器组成。将蛋白质混合物应用于一维阴离子交换柱，通过逐步增加氯化钠浓度进行洗脱分离。洗脱液直接引入二维反相柱，通过逐步增加乙腈浓度进行梯度洗脱进一步分离。两个洗脱阶段由计算机程序同步。通过该系统，非常复杂的蛋白质混合物，如粗制小脑提取物，可在12小时内可重复地分离为约200个峰。该方法可用于各种组织和细胞中蛋白质的全面分析，无需对样品进行复杂的预处理，并且允许对通过色谱法分离的蛋白质进行同时分析。分离出的蛋白质最适合用于蛋白质和基因序列分析策略。
