The objective of this Phase I proposal is to demonstrate the performance of a novel microfluidic device that will improve by a factor of 100 upon the performance of traditional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) used to separation of complex protein mixtures. Specific goals are to achieve a device peak capacity of 25,000 (compared to 3,000 for 2D-PAGE) and a cycle time of 1.5 hours (compared to 3 days for 2D-PAGE). The new device also improves by a factor ot ten on the peak capacities of existing 2D chromatography, capillary electrophoresis and microfluidic devices;the cycle time is roughly comparable. The new device is interfaced to ESI mass spectrometry, enabling it to determine top-down protein expression profiles. The proposed microfluidic device combines capillary isoelectric focusing (cIEF) with a single fast capillary zone electrophoresis (CZE). Careful attention is paid to reducing primary and secondary causes of resolution loss by suppressing electro-osmotic flow during focusing, preserving focused cIEF peak shapes when those peaks are mobilized, and careful coupling of cIEF with CZE. CZE flow rates are selected for large values. Separated proteins are then be removed through an ESI interface to a mass spectrometer. Proposal activities focus on demonstrating the key steps to demonstrate device feasibility. The device will be fabricated primarily at Arizona State University's Nanofab;process optimization will be used to create materials that are transparent and have other desirable physical properties. The device pores will be derivatized with high-density polyacrylamide to minimize protein adsorption. Performance of cIEF and CZE separations will be quantified using reference protein mixtures over a range of system operating conditions. Focused or separated proteins will further be mobilized from their separation regions to determine how peaks spread during device coupling. 2D separations will be studied, from known proteins mixtures to an E. coli lysate extract. Finally, device performance is verified by combining it with ESI mass spec. The device should find broad application to target discovery, determining the mechanism and progression of human disease, finding the activity of pharmaceutical compounds to known targets or the toxicity of these compounds to human organs, and identifying protein diagnostic markers.
Determining what proteins a cell or tissue expresses is a powerful complement to DNA analysis. Many human diseases are caused when abnormal proteins are produced, including enzymes, antibodies and hormones, and measuring these abnormal proteins directly is extremely helpful to their diagnosis and to creating of new pharmaceuticals. The tools developed in this proposal will determine and quantify with great precision and speed the proteins that are expressed in a format that is convenient to use. Its performance will improve on the current best technology by increasing by ten times the number of proteins that can be determined from one experiment, and reduce the analysis time by a factor of ten. The new tools should find broad application to target discovery, determining the mechanism and progression of human disease, finding the activity of pharmaceutical compounds to known targets or the toxicity of these compounds to human organs, and identifying protein diagnostic markers.
