Characterization of protein expression in breast cancer tissues by one and two-dimensional capillary electrophoresis.

摘要

Capillary electrophoresis (CE) coupled with laser-induced fluorescence detection can be used to produce high efficiency, high resolution, rapid, and automatable separations of bioanalytes at exceptional detection limits. One and two-dimensional CE separations were used to characterize the expressed protein fingerprints from both MCF-7 cellular homogenates and single MCF-7 breast cancer cells. While one-dimensional (1D) CE can resolve dozens of proteins, two-dimensional (2D) CE has the potential to resolve hundreds or thousands of proteins with broad dynamic range.; Proteins were derivatized with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), and detected by laser-induced fluorescence in a sheath-flow cuvette. The instrument was initially characterized and optimized using standard proteins. The limit of detection for this system was determined to be 5.8 zmol, based on the analysis of the carbonic anhydrase standard protein.; The chemical lysis of MCF-7 cells using detergents was optimized. MCF-7 cellular homogenates were separated by 1D-CE using the capillary sieving electrophoresis (CSE), capillary zone electrophoresis (CZE), and micellar electrokinetic capillary chromatography (MECC) modes. By hyphenating two of these 1D separation modes together in a 2D system, the peak capacity increased significantly for the analysis of MCF-7 cellular homogenates. A system hyphenating CSE and MECC was principally used. Throughput was increased dramatically by decreasing the length and inner diameters of separation capillaries and by increasing the electric field strength during analysis. Initial two-dimensional CE experiments took six or more hours to complete. After these changes were made, experiments were completed within one hour.; In this dissertation, the development of methods for single-cell analysis by 2D-CE is described. It is also demonstrated that by examining protein expression on a cell-to-cell basis, more information can be gained for a given tissue sample. Using this method, heterogeneous tissue samples can thus be better characterized and valuable information regarding low abundance cells within the cellular population can be gained. Variation associated with cell cycle-dependent protein expression was also investigated using the MCF-7 cell line. Additional work was done to characterize protein expression in single mouse embryos.