Analysis of cellular release using capillary electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.
In order to increase our understanding of the mechanisms of learning and memory in the central nervous system, it is necessary to know the neurotransmitters and neuromodulators used in the specific neuronal circuits under study. Methods have been developed to identify the peptides released from single neurons and neuronal clusters from the common neuronal model Aplysia californica. Specifically, solid-phase extraction (SPE), capillary electrophoresis (CE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) are combined for profiling neuropeptide releasates. A variety of combinations of SPE and CE were coupled off-line with MALDI-TOF-MS to reduce the high physiological salts, to concentrate the analytes, and to reduce the complexity of the mass spectra using separation. With these protocols, peptides and proteins up to 11000 Da were detected in releasates, offering a much wider mass range compared to direct MALDI analysis of the same releasates. A number of expected and unknown neuropeptides, including egg-laying hormone (ELH) and the partially processed delta/gamma-bag cell peptide were observed in the SPE-treated releasates from a single Aplysia-cultured bag cell neuron. However, by adding a CE separation after the SPE step preceding off-line MALDI-TOF-MS detection, the most complete neuropeptide profiles were obtained.
Rubakhin
S S
SS
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana 61801, USA.
Page
J S
JS
Monroe
B R
BR
Sweedler
J V
JV
D000818:Animals; D001048:Aplysia; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods; D004594:Electrophysiology / Q000379:methods; D017952:Ganglia, Invertebrate / Q000502:physiology*; D009479:Neuropeptides / Q000032:analysis*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D009479:Neuropeptides