Compositional analysis of small peptides using capillary electrophoresis and Ru(bpy)33+-based chemiluminescence detection

Ru(bpy)(3)(3+)-based chemiluminescence detection of underivatized amino acids was coupled to capillary electrophoresis and evaluated as a separation and detection methodology for the compositional analysis of peptides and proteins. Two tripeptides were studied: glycine-phenylalanine-alanine, and valine-proline-leucine, and the method was demonstrated to be quantitative and reproducible at the 10-mu g and 5-mu g peptide levels, with a limit of detection (LOD) of 2.5 pmol for gly-phe-ala and 80 fmol for val-pro-leu (S/N = 2), Ru(bpy)(3)(3+)-based chemiluminescence detection coupled to CE offers a sensitive and time-efficient method for the determination of the amino acid composition of peptides or proteins without derivatization. The detection limits for most amino acids were demonstrated to be at, or below the level achievable using ninhydrin or phenyl isothiocyanate derivatization. Amino acids were identified by their migration time through the capillary and their relative luminescence. The relative luminescent response obtained upon reaction of Ru(bpy)(3)(3+) with amino acids was dependent upon the amino acid R-group at the alpha-carbon, and the relative response may be useful in broadly classifying a number of amino acids. (C) 2000 Elsevier Science B.V. All rights reserved.