Development of high-performance techniques for the separation and analysis of minor protein components in human plasma

在分析人血浆和淋巴细胞中总蛋白的过程中，我们使用了二维聚丙烯酰胺凝胶电泳（2-D PAGE），因为它具有最佳的蛋白质分析分辨率。然而，大多数总蛋白分析工作者都是在变性条件下使用二维PAGE，以获得在二维凝胶上分离的多肽的结构信息。然而，蛋白质分析的最终目的必须是重建蛋白质在活细胞或器官中的功能。为此，我们一直在使用无变性剂的2-D PAGE。在本研究项目中，我们提出了四种二维PAGE技术（Type-I, Type-II， Type-III和Type-IV）的组合，将多肽的结构信息（通过Type-IV二维PAGE获得）与蛋白质的功能信息（通过Type-I二维PAGE获得）相关联。此外，我们还开发了新的蛋白质分离和回收电泳技术，克服了传统二维PAGE技术的不足。在常规的2-D PAGE中，电泳后的蛋白质固定在平板凝胶上，用染料或硝酸银染色，用安装在微型计算机上的图像分析软件定量，并从凝胶片中提取进一步的结构分析。这些程序大多需要人工操作，几乎不可能实现自动化。在1996-1998年期间，我们开发了毛细管电泳分离蛋白质的技术。毛细管电泳的特点是柱上检测，柱上定量，和全自动化的分离过程。建立了毛细管等电聚焦和毛细管SDS电泳条件，并将其应用于人血浆蛋白的分离。然后，我们扩展了毛细管电泳的条件，旨在建立高效的蛋白质分离和回收技术。我们已经为此目的建造了一些仪器和装置，我们正在评估它们在分离人血浆中微量蛋白质成分方面的性能。少

In the course to analyze total proteins in human plasma and lymphocytes, we have used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) since it has the best resolution for the analysis of proteins. However, most of the workers on total protein analysis are using 2-D PAGE in denaturing conditions, to obtain structural information of the polypeptides separated on the 2-D gels. However, ultimate aim of protein analysis must be the re-construction of protein functions in living cells or organs. For this purpose, we have been using 2-D PAGE in the absence of denaturants. In this research project, we proposed a combination of four 2-D PAGE techniques (Type-I, Type-II, Type-III and Type-IV) to correlate structural information of polypeptides (obtained by Type-IV 2-D PAGE) with information on protein functions (obtained by Type-I 2-D PAGE).Further, we developed new electrophoretic techniques of protein separation and recovery which overcome the shortage of conventional 2-D PAGE te … More chniques. In conventional 2-D PAGE, after electrophoresis proteins are fixed on the slab gel, stained with a dye or silver nitrate, quantitated by an image analysis software installed in a microcomputer, and extracted from the gel piece for further structural analysis. Most of these procedures require manual operation and their automation is almost impossible. During 1996-1998, we have developed techniques of capillary electrophoresis for the separation of proteins. Capillary electrophoresis is characterized by on-column detection, on-column quantitation, and full-automation of the separation process. We established the conditions of capillary isoelectric focusing and capillary SDS electrophoresis and applied them for the separation of human plasma proteins. Then we extended these conditions of capillary electrophoresis aiming to establish high-performance techniques for protein separation and recovery. We have constructed several apparatus and devises for this purpose and we are evaluating their performance in the separation of minor protein components in human plasma. Less