For complex protein mixtures such as cell lysates or sera containing thousands of proteins, 2-D polyacrylamide gel-electrophoresis (2D-PAGE) is the standard method used to isolate individual low-abundance proteins for identification and quantitative analysis. However, it is a tedious and time-consuming manual procedure. Alternative separation methods that can be automated typically involve chromatographic and mass spectral procedures that do not have the separation capabilities of 2D-PAGE or are not easily used for quantitative analysis. A platform that has the separation efficiency of 2D-PAGE, is rapid (less than several hours), and easily automated is needed for quantitative analysis of clinical samples. LCM Technologies is currently developing a capillary analog to the standard 2D-PAGE method that performs iso-electric focusing (IEF) on a micro-fluidic chip in a channel a few centimeters long, after which IEF segments are mobilized for injection into a capillary array for SDS capillary array electrophoresis (SDS-CAE) with laser-induced fluorescence detection (LIF) and MALDI-TOF mass spectral analysis. Once completed, the system will be fully automated with a total separation time of less than two hours and is expected to have separation efficiency comparable to or better than 2D-PAGE. LIF detection will allow quantitative measurement of separated proteins with a dynamic range of four orders-of-magnitude. It also allows multiple dyes to be used for simultaneous detection of size and pI markers for reproducible measurement of specific proteins. The main innovation is a scheme to couple microchip IEF to the capillary array for SDS-CAE with high efficiency and with quantitative transfer. The system will be characterized for efficiency across the array and run-to-run, which is required for quantitative analysis. In addition, fluorescence detection sensitivity will be characterized to determine detection limits in the original sample, which are expected to be better than 1 ng/mL for individual proteins. The relevance to public health is that the technology will be developed for rapid and accurate measurement of cancer and other disease markers in serum and other bodily fluids that has the capability of early. This could be used for routine screening and monitoring for cancer and other diseases in patients, which will allow early treatment to increase survival rate. ? ? ?
