|Functional Genomics Section
DOE Human Genome Program Contractor-Grantee Workshop
144. Microfabricated Microfluidic Devices for Proteome Mapping
R.S. Ramsey, R.S. Foote, R.D. Rocklin,
M.I. Lazar, Y. Liu, and J. M. Ramsey
Miniaturized chemical instruments, "Lab-on-a-Chip" technologies, are being developed for rapid, comprehensive analysis of cellular proteins, as an alternative to the slow and labor-intensive 2D gel methods currently used for protein mapping. The microfabricated devices will integrate on a single structure, elements that enable multidimensional separations of protein mixtures and electrospray ionization of the analytes for direct, on-line interfacing with mass spectrometry. The platform exploits the many advantages of Lab-on-a-Chip devices, including small size, inexpensive fabrication, high speed, low volume materials consumption, high throughput, and automated operation. Potential applications of the technology include quantification of gene product levels in specific cell types, comparative analysis of patterns of gene expression in different tissues at different stages of development, analysis of structural and/or expression level changes resulting from mutagenesis or genetic disease, and identification of specific protein markers of disease.
The conventional 2D PAGE method for resolving cellular proteins is not only laborious but also has poor reproducibility, sensitivity, and sample recovery. Individual spots may be identified off-line using mass spectrometry (MS) but sample extraction and transfer processes are inefficient. Column liquid chromatography or capillary electrophoresis (CE), which are more easily coupled with MS using electrospray ionization (ES), in general, lack the resolution required for the analysis of complex biological samples. Two-dimensional separations greatly increase the resolving power, provided the individual methods are orthogonal, and when combined with MS result in a powerful technique, given the multiplicative effect of joining different separation mechanisms. We have designed and demonstrated an integrated device combining micellar electrokinetic chromatography and high-speed free zone electrophoresis. The orthogonality of these techniques, an important factor for maximizing peak capacity or resolution elements, was verified by examining each technique independently for peptide separations. The two dimensional separation strategy was found to greatly increase the resolving power over that obtained for either dimension alone. The integrated device operates by rapidly sampling and analyzing effluent in the second dimension from the first dimension. Second dimension analyses are performed and completed every few seconds. Total analysis times are less than 10 min and the peak capacity has been estimated to be in the 500 to 1000 range. The operation of the device is completely automated. Microchips have also been interfaced to a time-of-flight mass spectrometer that has acquisition rates necessary to capture mass spectra from rapidly eluting components. The electrospray element will eventually be integrated with the two dimensional separations to allow on-chip MS analysis for ultra-high throughput protein mapping. Increases in sample throughput are anticipated to be greater than two orders of magnitude as compared to 2D PAGE.
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