Peptide analysis using capillary electrophoresis with fluorescence detection

The study of amino acids and peptides in biological samples requires analytical methods and instrumentation capable of providing highly sensitive data about analytes in complex matrices. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) provides the combination of highly efficient separations with sensitive optical detection. The application of CE-LIF for the analysis of amino acids and peptides is reviewed.; To provide additional chemical information on analytes, wavelength resolution can be employed in CE-LIF. Through the use of imaging spectrographs and array detectors it is possible to acquire full emission spectra during the course of a separation. Spectral information is useful for analyte classification and spectral deconvolution of co-migrating species. A description of the instrumentation and data handling techniques used in wavelength-resolved CE-LIF is presented along with an application of spectral deconvolution for studying bilirubins in human serum.; It is possible to analyze amino acids and peptides a number of different ways using CE-LIF, including pre-capillary derivatization, post-capillary derivatization and native fluorescence. While pre-capillary derivatization provides high sensitivity, problems can result due to inconsistent tagging and product stability. These can be avoided through the use of post-capillary derivatization. This technique involves mixing reagents with the analytes after they have been separated by CE. By adding o-phthaldialdehyde (OPA) and {dollar}beta{dollar}-mercaptoethanol to the buffer used in the sheath flow detection cell, this can be accomplished using the wavelength-resolved CE-LIF instrument. The system is characterized for a series of amino acids and the results are compared to those obtained using pre-capillary derivatization.; Since the amino acids tryptophan, tyrosine and phenylalanine contain aromatic functionalities, they can be detected without derivatization when the proper ultraviolet excitation wavelengths are used. The application of wavelength-resolved CE-LIF for the native fluorescence detection of these amino acids and peptides that contain them is described for the classification of peptides based on their spectra. Preliminary results are presented for combining this method with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for the identification of unknown peptides.