Development of a Capillary Electrophoresis (CE)-based Integrated Analytical Platform for Low Abundance Proteins

蛋白质组学是对生物系统中蛋白质的大规模研究，它导致了对人类生理学理解的范式转变和药物发现的进展。质谱学是蛋白质组学研究中的重要工具。在过去的十年中，MS的进步导致了速度、可靠性以及最重要的灵敏度的显著提高。在发现可能成为关键药物靶点的低丰度蛋白质时，敏感性至关重要。虽然今天的MS技术能够检测到非常少量的蛋白质，但目前大多数现有的样品制备程序需要更多的蛋白质。为了充分利用先进的MS的力量，以非常小的体积处理少量的蛋白质是必不可少的。 本文提出的研究重点是基于一种称为毛细管电泳法(CE)的先进分析技术，开发一种小体积、亚微升的样品处理平台。杨家诚此前曾演示过使用毛细管电泳法对微量蛋白质进行一些样品制备反应。在这一应用中，他建议使用磁性微珠将这些单独的反应整合在一起，以极小的数量传输分析物。这样一个集成的分析平台将使整个多步骤样品制备能够在微型水平上按顺序进行。这一结果有望在样本量严重受限的情况下进行超灵敏的蛋白质分析。 为了说明其对社会的好处，将在两个领域应用所开发的方法。第一种是牙齿表面形成的蛋白质膜的分析。这种膜的成分直接影响与龋齿和牙周感染相关的口腔微生物活动。第二个应用重点是蛋白激酶CK2，它在癌细胞中的水平普遍升高。这些新方法将用于研究CK2在细胞程序性死亡中的抑制作用，即CK2‘S在癌细胞中的作用。在新开发的技术的推动下，这两项研究都将在预防和治疗这些疾病方面立即取得进展。

Proteomics, a large-scale study of proteins in biological systems, led to a paradigm shift in the understanding of human physiology and drug discovery advances. Mass spectrometry (MS) is an essential tool in proteomics. Advancements in MS over the past decade have resulted in dramatic improvements in speed, reliability and most importantly, sensitivity. Sensitivity is vital in the discovery of low abundance proteins that can be crucial drug targets. While today's MS technology is capable of detecting very small quantities of proteins, most of the currently existing sample preparation procedures require much larger amount of proteins. To fully utilize the power of advanced MS, handling of small quantities of proteins in very small volumes is essential. The research proposed herein focuses on the development of a small-volume, sub-microliter, sample-handling platform based on an advanced analytical technique called capillary electrophoresis (CE). Yeung has previously demonstrated the use of CE to perform a number of sample preparation reactions for minuscule quantities of proteins. In this application, he proposes the integration of these individual reactions using magnetic microbeads for analyte transport at minuscule quantities. Such an integrated analytical platform will allow the entire multiple-step sample preparation to be conducted in sequence at a miniaturized level. The outcome is expected to enable ultrasensitive protein analysis when sample quantity is severely limited. To illustrate its benefits to society, the developed methodology will be applied in two areas. The first one is analysis of the protein film formed on tooth surfaces. The composition of this film directly affects the oral microbial activities related to tooth decay and periodontal infection. The second application focuses on protein kinase CK2, which is commonly found at elevated levels in cancer cells. The proposed new methods will be applied to study the inhibiting effects of CK2 in programmed cell death; i.e., CK2's role in cancer cells. Both of these studies, enabled by the newly developed techniques, will lead to immediate advances in prevention and treatment of these diseases.