Liquid Chromatography of Biological Substances

本研究旨在进一步扩大现代液相色谱的应用范围，以满足医学和生命科学对高速、高分辨率分离方法的需求。在蛋白质组学框架内复杂蛋白质混合物研究的最新进展产生了这种需要。我们的主要研究重点是建立对毛细管电色谱（CEC）分离中迁移过程的物理化学基础的坚实理论认识，这是一种具有巨大潜力的新型分离技术。这种新的高效分离方法可以看作是色谱和电泳的混合。换句话说，我们可以把CEC看作是电泳平台上的色谱，这体现了两种主要生物分析技术的优势。CEC采用小内径的毛细管柱，在20-125 μ m范围内，用高电场驱动电氧化流动相流。它可以帮助实现非常高的色谱柱效率。我们对CEC理论和应用的基础研究有望支持我们计划在实验室量身定制的新固定相的设计，制备和使用。在许多方面，与CEC开发相关的技术问题与基于芯片的分析系统开发中遇到的问题非常相似。毛细管和芯片都代表了分析仪器小型化的趋势。因此，毛细管柱研究的进展有望有利于基于芯片的集成分析系统的发展。我们的这一部分研究将极大地受益于我们在过去几十年里为HPLC制备和表征固定相所获得的经验。我们的理论研究结果以及我们在色谱柱工程和CEC方面的经验将用于计划研究的第三部分：蛋白质组学。它代表了蛋白质化学的一个新领域，一旦成功，它有望对各种疾病的诊断方式产生重大影响。我们的努力将得到耶鲁大学人类遗传学教职员工和耶鲁大学凯克核心设施的支持。我们将从改进一种被称为ICAT技术的有前途的分析方法的分离步骤开始。此外，我们计划通过提高温度来提高这种方法的分析速度。最后，我们计划通过采用置换色谱步骤来提高该技术的灵敏度。在该程序的更高级的开发步骤中，我们将设计一个半自动化，甚至是全自动的蛋白质组学分析仪。它应具有较高的分离效率，有利于加快蛋白质组学和蛋白质层析的分析分离。

This proposal is aimed at further expanding the scope of modem liquid chromatography in a major way to meet the needs in medicine and life sciences for separation methods of high speed and resolution. The need is engendered by recent advances in the study of complex protein mixtures within the framework of proteomics. The main focus of our research is the establishment of a solid theoretical understanding of the physico-chemical underpinning of the migration processes that are involved in the separation by capillary electrochromatography (CEC), a novel separation technique of great potential. This new high performance separation method can be considered to be a hybrid of chromatography and electrophoresis. In other words, we can look at CEC as chromatography on a electrophoretic platform, that embodies the advantages of the two leading bioanalytical techniques. CEC employs capillary columns of small inner- diameter, in the range of 20-125 mum, and a high electric field to drive the mobile phase flow by electrosmosis. It can facilitate the realization of extraordinary high column efficiencies. Our fundamental studies on the theory and application of CEC are expected to support the design, preparation and use of the new stationary phases we plan to tailor make in our laboratory. In many respects the technical problems associated with the development of CEC are very similar to those encountered in the development of chip based analytical systems. Both capillaries and chips represent the trend of miniaturization of analytical instrumentation. Therefore advancements in our research with capillary columns are expected to benefit the development of chip based integrated analytical systems as well. This part of our study would greatly benefit from the experience we have gained with preparing and characterizing stationary phases made in our laboratory for HPLC over the last decades. The results of our theoretical studies together with our experience in column engineering and CEC will be utilized in embarking on the third part of the planned investigation: proteomics. It represents a new area of protein chemistry and upon success it is expected to have a great impact on the way in which various illnesses will be diagnosed. Our endeavor will be supported by Yale faculty members in human genetics and in the Keck core facility at Yale. We shall start with improving the separation steps of a promising analytical method referred to as ICAT technology. Further we plan to enhance the speed of analysis of this approach by using elevated temperature. Lastly, we plan to increase the sensitivity of the technique by employing a displacer chromatographic step. In the more advanced development steps of this procedure we shall design a semi, or even, a fully automated proteomic analyzer. It should have high separating efficiency and facilitate speeding up the analytical separation in proteomics and in protein chromatography at large.