| This dissertation addressed several issues concerning selective fluorescent derivatization of peptides for separation and characterization by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The central theme of this research was the development of instrumentation and derivatization methods which were suitable for peptide mapping applications.; A LIF detector was developed to scan the entire length of the separation capillary. The scanner was based on epi-illumination of the capillary with a confocal optical emission arrangement. The system design and signal optimization are described in the first manuscript. Applications of the system are also presented and include detection, without mobilization, of fluorescently labeled proteins separated by isoelectric focusing and measurement of solute diffusion coefficient.; Two site-specific fluorescent derivatization schemes were examined next for the purpose of peptide labeling. In the second manuscript, a method for blocking the amine groups of phosphorylated β-casein by reductive dimethylation is presented. The derivatized protein was next allowed to digest with trypsin or CNBr. The resulting peptides were labeled with fluorescein isothiocyanate and separated by CE with LIF detection. The resulting peptide maps were demonstrated to be highly reproducible and generally insensitive to fluorescent labeling conditions.; In the final manuscript, a second approach is described in which the amine groups of insulin B-chain are temporarily blocked by reductive dihydroxypropylation. Following tryptic cleavage, the peptides were labeled with 6-(fluorescein-5-carboxamido)-2-phenylethyl chloromethyl ketone. The dihydroxypropylated amine derivatives contained a substituted α-amino alcoholic function and were susceptible to periodate oxidation to regenerate the native protein. Therefore, the final product was a set of native peptides that each contained a single fluorescent label at the N-terminal amino group, which were analyzed by CE with LIF detection. Matrix-assisted laser desorption time-of-flight mass spectrometry was also employed to ensure completeness of the reactions and to confirm the identity of the tryptic peptides. |
