Analysis of trace level peptides using capillary electrophoresis with UV laser-induced fluorescence

Abstract

In order to measure trace level peptides (< 1 μM) in exceedingly small sample sizes (< 100 pl) from neuronal or subneuronal samples, sophisticated separation and detection methods are required. Novel instrumentation is described that employs UV laser excitation of fluorescamine labeled peptides for fluorescence detection in capillary electrophoresis. Small injections of standard analyte mixtures or biological samples are efficiently separated on the basis of their electrophoretic mobilities in an electric field applied across the capillary. For sensitive detection, we use the 354 nm line from a helium-cadmium laser for excitation, a reflecting microscope objective for fluorescence collection, and a photomultiplier tube for photon counting. A large excess of fluorescamine derivatizes amino acids and peptides down to concentrations of 10 nM with a previously unreported but manageable fluorescence background resulting from reagent hydrolysis and impurities. This detection method gives a linear working range of four orders of magnitude and a limit of detection for labeled Aplysia californica egg-laying hormone of 2.3 × 10 −9 M. Finally, assays of individual Aplysia c. neurons produce electropherograms unique to the identified neuron type and peaks corresponding to peptide standards.