Native Fluorescence Detection and Spectral Differentiation of Peptides Containing Tryptophan and Tyrosine in Capillary Electrophoresis

A native fluorescence detection system for capillary electrophoresis is described that achieves low attomole detection limits and simultaneous acquisition of complete fluorescence emission spectra. The system is designed for detection of peptides through the intrinsic fluorescence of tryptophan and tyrosine residues. The detection system employs a frequency doubled krypton laser operating at 284 nm for excitation, a sheath flow cell, a reflective f/1.2 microscope objective, an imaging spectrograph, and a CCD detector. The detection capabilities are characterized with tryptophan and tyrosine, which have limits of detection (3σ) of 2 × 10^-10 and, 2 × 10^-8 M, respectively. Acquisition of the fluorescence emission spectrum provides the ability to distinguish three classes of peptides: those that contain tryptophan, those that contain tyrosine, and those that contain both tryptophan and tyrosine.