Neurotransmitter sampling and storage for capillary electrophoresis analysis.

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleiuohbrain-Chae californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at -20 degrees C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites.

Measuring the peptides in individual organelles with mass spectrometry.

New sampling protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allow the assay of single dense core vesicles. Understanding the packaging of vesicles is important as vesicles are the quanta of information for intercellular communication. Using vesicles from the exocrine atrial gland of Aplysia californica as the model, a wide range of bioactive peptides are detected within each vesicle. Although the expression of the egg-laying hormone gene family of type 1 atrial gland cells has been previously examined, chemical characterization of individual 1-2 microm diameter vesicles demonstrates that products from several genes are colocalized. The mass sensitivity of MALDI MS can be further improved to enable the analysis of even smaller subcellular organelles.

Factors affecting quantitative electrokinetic injections from submicroliter conductive vials in capillary electrophoresis.

The factors influencing quantitative electrokinetic injections in capillary electrophoresis for custom 340-nL, 10-microL, and 110-microL stainless steel sample vials have been investigated using a six-analyte mixture containing catecholamines and indolamines. Deleterious sample degradation is increased with smaller sampling vials, decreased capillary-electrode distances, and increased current passed during the injection. Zero-voltage injections from the smallest vials also demonstrate additional injection discrepancies when compared to larger-volume bulk solution injections. These effects are in addition to the electrokinetic bias and complicate the selection of appropriate internal standards. For nanoliter-volume conductive vials, the injection process creates new species and eliminates other electroactive species to such an extent that quantitation becomes problematic.

Single neuron analysis by capillary electrophoresis with fluorescence spectroscopy.

A technique to identify and quantitate simultaneously more than 30 compounds in individual neurons is described. The method uses nanoliter volume sampling, capillary electrophoresis separation, and wavelength-resolved native fluorescence detection. Limits of detection (LODs) range from the low attomole to the femtomole range, with 5-hydroxytryptamine (or serotonin [5-HT]) LODs being approximately 20 attomoles. Although the cellular sample matrix is chemically complex, the combination of electrophoretic migration time and fluorescence spectral information allows positive identification of aromatic monoamines, aromatic amino acids and peptides containing them, flavins, adenosine- and guanosine-nucleotide analogs, and other fluorescent compounds. Individual identified neurons from Aplysia californica and Pleurobranchaea californica are used to demonstrate the applicability and figures of merit of this technique.

Characterizing submicron vesicles with wavelength-resolved fluorescence in flow cytometry.

Individual synthetic vesicles 0.1-1.0 micron in diameter are sized by using single-particle fluorescence emission spectra obtained in a custom sheath flow cell with an imaging spectrograph and a charge-coupled device. Data are acquired at 1 Hz, with limits of detection (3 sigma) less than 6.0 x 10(3) and 1.0 x 10(4) molecules of free sulforhodamine 101 and fluorescein, respectively, and with a spectral range for fluorescence emission collection from 350 to 800 nm (0.45 nm/pixel resolution). The system is used for small-particle population analysis by analyzing a suspension of submicron, unilamellar, synthetic vesicles prepared by standard procedures with phosphatidylserine and Texas red- or fluorescein head-group-conjugated dihydropalmitoylphosphatidylethanolamine. The submicron particles are individually identified, sized, and discriminated based on single-particle fluorescence emission spectra. Excellent agreement is found between fluorescence sizing data and transmission electron microscopic measurements.