Development of Dynamic Isoelectric Focusing for Cancer Proteomics

描述（由申请人提供）：虽然在癌症DNA分析领域取得了很大的进展，但了解DNA编码的蛋白质可以提供更多的答案，但也更具挑战性。随着癌症蛋白质组学研究的进展，很明显，需要新的分析工具和技术来全面分析细胞中的蛋白质，以便我们对癌变的理解以及健康细胞和癌细胞之间的差异可以取得进展。对癌症蛋白质组学的进一步了解将推动新的药物靶点的发现，因为在没有先入为主的想法的情况下，观察细胞中的分子变化是最有价值的。由于细胞中蛋白质的数量非常多，综合分析需要使用具有高峰值容量的分离方法。毛细管等电聚焦（cIEF）在这一领域显示出巨大的前景，其峰值容量超过1400。这大大超过了传统的分离方法，如液相色谱（LC）、毛细管电泳（CE）或质谱（MS），这些方法的峰值容量通常小于200。当多种分离技术相结合时，可以实现系统总峰值容量的增加，从而导致串联方法（如LC/LC或LC/MS）的普及和性能。虽然cIEF优于CE和LC的性能似乎使其成为串联系统的首选，但它无法与其他方法有效地接口。这是它没有被广泛使用的主要原因。该研究将继续发展动态等电聚焦技术，这是一项由PI开发的新技术，将能够提供cIEF的高峰值容量，同时还能有效地与其他技术耦合。这种组合系统将很容易超越其他串联方法，并将对癌症的分子分析产生重大影响，因为它们将允许获得比目前可能的更全面的癌细胞蛋白质概况。通过将动态IEF与MALDI-MS结合，并使用该系统分析和观察处理过和未处理过的PC-3前列腺癌细胞提取物的差异，将证明动态IEF的能力。细胞治疗将基于Co-PI目前研究的化合物，如双脱氢羟基乙酸，这是一种雌激素羧酸，被证明能有效减少前列腺癌的增殖。

DESCRIPTION (provided by applicant): Though great progress has been made in the area of DNA analysis for cancer, understanding the proteins encoded by DNA can provide more answers, but is also more challenging. As the study of cancer proteomics advances, it is clear that new analytical tools and technology are needed for the comprehensive profiling of the proteins in a cell so that our understanding of carcinogenesis and the differences between healthy and cancerous cells can progress. Further understanding of cancer proteomics will drive the discovery of new drug targets as molecular changes in the cell are observed without preconceived ideas about what changes would be the most valuable to monitor. Due to the very large number of proteins in a cell, comprehensive analyses require the use of separation methods that have high peak capacities. Capillary isoelectric focusing (cIEF) has shown great promise in this area with a peak capacity in excess of 1400. This greatly exceeds traditional separation methods, such as liquid chromatography (LC), capillary electrophoresis (CE), or mass spectrometry (MS), which often have peak capacities of less than 200. An increase in the total peak capacity of a system can be achieved when multiple separation techniques are combined, leading to the popularity and performance of tandem methods such as LC/LC or LC/MS. Though the superior performance of cIEF over CE and LC would seem to make it a preferred choice in a tandem system, it is not able to be efficiently interfaced with other methods. This is the primary reason it is not widely used. The proposed research will continue the development of dynamic isoelectric focusing, which is a new technology developed by the PI that will be able to provide the high peak capacity of cIEF while also efficiently coupling with other techniques. The combined systems made possible will easily outperform other tandem methods and will have a high impact on the molecular analysis of cancer because they will permit the acquisition of a more comprehensive profile of the proteins in cancerous cells than is currently possible. The capabilities of dynamic IEF will be demonstrated by interfacing it to MALDI-MS and using the system to analyze and observe differences in extracts from treated and untreated PC-3 prostate cancer cells. The cell treatment will be based on compounds currently researched by the Co-PI, such as bisdehydrodoisynolic acid, which is an estrogenic carboxylic acid shown to be effective at reducing the proliferation of prostate cancer.