Isoelectric point analysis of proteins and peptides by capillary isoelectric focusing with two-wavelength laser-induced fluorescence detection

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection can be a highly effective tool for analysis and characterization of proteins and peptides. Here we present a two-wavelength LIF detection system for the capillary isoelectric focusing (cIEF) of proteins and peptides. The helium-neon laser (543.5 nm) was used for simultaneous excitation of biomolecules labeled with two different fluorophores. The fluorescent emission signal from the different fluorophores could then be separated by a dichroic mirror, spectrally filtered, and collected in discrete channels. The system performance characteristics, such as spectral overlap in the channels and selection of fluorescent reporters, are presented here. Finally, we demonstrate applications of this system through protein and peptide analysis. These included the use of isoelectric point markers, each labeled with a fluorescent reporter moiety. Co-injection of the standards with the zwitterion to be determined provided for in-run calibration of pH gradients and for internal correction of variations in migration time.