Analysis of Barrett's esophageal tissues by two-dimensional capillary electrophoresis: An investigation of coupling different separation methods.

摘要

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) is used to separate and detect biomolecules in complex mixtures, such as cellular homogenates and single cells. One and two-dimensional CE separations were used to obtain protein profiles from Barrett's esophageal homogenates and single cells.;Several different separation mechanisms were investigated in one and two-dimensional CE systems. These included capillary zone electrophoresis, capillary sieving electrophoresis, micellar electrokinetic chromatography, and capillary isoelectric focusing. Two-dimensional CE systems involved hyphenating CSE and MEKC, as well as clEF and C7F. These two 2D CE systems were compared for the analysis of standard proteins and Barrett's esophageal homogenates.;2D CSE-MEKC was used to characterize biomolecules in Barrett's esophageal homogenates and single cells. Biomolecules are labeled by 3-(2-fluoryl)quinonoline-2-carboxaldehyde (FQ), a fluorogenic molecule that binds to primary amines to form highly fluorescent products. Fluorescent biomolecules are separated by 2D-CE and detected inside a sheath-flow cuvette with a 473 nm solid state laser and fiber-coupled avalanche photo diode (APD). 2D-CE-LIF is capable of detecting zeptomole (10 -21) quantities of FQ-labeled proteins. Biopsies from patients with Barrett's esophagus were treated with EDTA and dissected into stroma tissue and epithelial tissue. The profiles obtained from the different tissue types were compared. Individual crypts were also dissected and analyzed by 2D CE. It was observed that smaller biomolecules were separated in the MEKC dimension with high efficiency; however, bigger protein molecules were not well resolved in the MEKC dimension.;Capillary isoelectric focusing and capillary zone electrophoresis were coupled with laser-induced fluorescence detection to create an improved two-dimensional separation method for proteins. In this method, two capillaries were joined through a buffer filled interface. Separate power supplies controlled the potential at the injection end of the first capillary and at the interface; the detector was held at ground potential. Proteins were labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The separation produced a spot capacity of over 500.