DEVELOPMENT OF DYNAMIC ISOELECTRIC FOCUSING FOR CANCER PROTEOMICS


Year of Award:
2006
Award Type:
R21
Project Number:
CA120691
RFA Number:
RFA-CA-06-002
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
TOLLEY, LUKE
Other PI or Project Leader:
N/A
Institution:
SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
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.