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

开发基于毛细管电泳 (CE) 的低丰度蛋白质集成分析平台

• 批准号: 250020-2012
• 负责人: Yeung, Ken
• 金额: $ 2.55万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=570793
• 关键词: Development; Capillary; Electrophoresis; CE; based

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.

蛋白质组学是对生物系统中蛋白质的大规模研究，导致了对人类生理学和药物发现进展的理解的范式转变。质谱（MS）是蛋白质组学中的重要工具。在过去的十年中，MS的进步导致了速度，可靠性和最重要的灵敏度的显着改善。灵敏度对于发现低丰度蛋白质至关重要，这些蛋白质可能是关键的药物靶点。虽然今天的MS技术能够检测非常少量的蛋白质，但大多数现有的样品制备程序需要大量的蛋白质。为了充分利用先进MS的能力，以非常小的体积处理少量蛋白质是必不可少的。 本文提出的研究重点是开发一个小体积，亚微升，样品处理平台的基础上，先进的分析技术，称为毛细管电泳（CE）。Yeung先前已经证明了使用CE对微量蛋白质进行许多样品制备反应。在这一应用中，他提出了使用磁性微珠将这些单独的反应整合起来，以极少量的方式进行分析物运输。这种集成的分析平台将允许整个多步骤样品制备以小型化水平依次进行。 该结果有望在样本量严重受限时实现超灵敏蛋白质分析。 为了说明其对社会的好处，开发的方法将应用于两个领域。第一个是牙齿表面蛋白膜的分析。这种薄膜的组成直接影响与蛀牙和牙周感染有关的口腔微生物活动。第二个应用集中在蛋白激酶CK 2，它通常在癌细胞中水平升高。所提出的新方法将用于研究CK 2在程序性细胞死亡中的抑制作用;即，CK 2在癌细胞中的作用这两项研究，通过新开发的技术，将导致这些疾病的预防和治疗的直接进展。