Subcellular analysis of D-aspartate.
D-Aspartate (D-Asp) is an especially intriguing molecule found within neurons of the central nervous system of animals ranging from mollusks to vertebrates. It has a large variety of roles ascribed to it, including an involvement in cell-to-cell signaling. To determine the D-Asp content in cells and in subcellular domains, a laboratory-assembled capillary electrophoresis system with laser-induced fluorescence (LIF) detection has been used. The system allows chiral separations with sufficient sensitivity and selectivity to measure the D-Asp content in specific subregions of a single neuron, including neuronal processes. The method uses microvial sampling, analyte derivatization with naphthalene-2,3-dicarboxaldehyde, cyclodextrin-mediated micellar electrokinetic capillary chromatography, and sheath flow cell-based LIF detection. Manipulating neuronal processes is difficult as they often disintegrate during the transfer to the sampling vial. We describe a glycerol treatment that stabilizes cell morphology during sample preparation, thereby alleviating the deleterious effects of the high-salt extracellular matrix on the electrophoretic separation. D-Asp percentages in processes from identified neurons from Aplysia californica differ significantly depending on the cell studied. Subcellular analysis reveals more compounds in the cell body than in the processes.
Miao
Hai
H
Department of Chemistry, University of Illinois at Urbana--Champaign, 61801, USA.
Rubakhin
Stanislav S
SS
Sweedler
Jonathan V
JV
D000818:Animals; D001048:Aplysia; D001224:Aspartic Acid / Q000032:analysis* / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism
D001224:Aspartic Acid