Two-Dimensional Capillary Electrophoresis for Analysis of Proteins

1 Instrument We reported the first two-dimensional capillary electrophoresis system for protein analysis (Michels et al., 2002, Hu et al., 2004, Michels 

The interface is constructed by macromachining grooves in a Plexiglas block. These grooves are about 1 mm wide and deep, and are milled using an appropriately sized bit. Two nested sets of tubes are placed within the capillary these tubes align the 

Two high-voltage power supplies are used to drive the separation. The first power supply applies high voltage through a platinum electrode to the injection buffer reservoir for the first dimension separation. The second power supply applies potential to the interface through its buffer reservoir. The sheath flow cuvette is held at ground potential. 

After this prerun, the voltage is programmed to periodically pulse a plug of analyte into the interface. This fraction is then drawn into the second capillary for further separation. In our current configuration, the separation window in the CSE dimension is roughly 200 s in duration, and roughly 200 pulses of 1 s duration are required for the contents of the CSE capillary to be transferred to the second dimension. A constant potential is applied across the second dimension capillary, typically 10,000-20,000 V. Under this constant voltage, any analyte present within the interface is driven into the second dimension capillary for separation. Detection is by laser-induced fluorescence in a postcolumn sheath-flow cuvette. 

2 Data Presentation The raw electrophoresis data consist of a long file of the intensity trace recorded by the fluorescence detector (Fig. 15.4). The trace consists of a pseudoperiodic set of peaks, where successive peaks are generated by a component, which is transferred in several fractions from the first to the second 

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