TY - JOUR
T1 - Subcellular analysis of D-Aspartate
AU - Miao, Hai
AU - Rubakhin, Stanislav
AU - Sweedler, Jonathan V
PY - 2005/11/15
Y1 - 2005/11/15
N2 - 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.
AB - 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.
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U2 - 10.1021/ac0511694
DO - 10.1021/ac0511694
M3 - Article
C2 - 16285665
AN - SCOPUS:27944511108
SN - 0003-2700
VL - 77
SP - 7190
EP - 7194
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 22
ER -