Separation of dynorphin peptides by capillary electrochromatography using a polydiallyldimethylammonium chloride gold...

Dynorphin A (Dyn A) is an endogenous opioid peptide found in blood and central nervous system tissue at very low concentrations. Elevated levels of Dyn A due to different disease states, for example neurodegenerative disease, have been linked to toxic nonopioid activity. CE is a powerful technique that can achieve high-efficiency separations of charged analytes. However, CE has limited use for the analysis of basic proteins and peptides, due to their adsorption onto the inner surface of the fused silica at pHs below their pI. This adsorption can lead to a loss of efficiency, irreproducibility of migration times, and peak tailing. To obviate this problem, a polydiallyldimethylammonium chloride-stabilized gold nanoparticle-coated capillary was investigated for the separation of dynorphin metabolites. The positively charged gold nanoparticles (GNP) minimized unwanted adsorption of the positively charged peptides onto the surface of the fused-silica capillary. Separation efficiency and resolution for opioid peptides Dyn A (1-6), Dyn A (1-7), Dyn A (1-8), Dyn A (1-11), and leu-enkephalin on the GNP-coated capillary column were evaluated under different experimental parameters. The best separation of Dyn A (1-17) and its fragments was achieved using a BGE that consists of 40 mM sodium acetate buffer (pH 5) containing 5% GNP, a field strength of -306 V/cm, and a 75 μm id capillary. The developed method was applied to the separation of tryptic peptide fragments of dynorphin A (1-17).