This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Synaptic transmission is facilitated by vesicular trafficking of neurotransmitters. The properties and components of synaptic vesicles have been increasingly studied. We are interested in two types of synaptic vesicles (SV), classic synaptic vesicles (CSV) and dense core vesicles (DCV). Studies from our laboratory and others have shown specific protein and neurochemical co-localization with both CSVs and DCVs relevant to neurophysiology and neuropathophysiology. Our studies utilize the tools of mass spectrometry and cell biology for a detailed proteomic study on the components of SVs using existing methodology such as capillary HPLC, ion exchange chromatography, tandem mass spectrometry, and immunohistochemistry. Early results using tandem mass spectral data of in-gel proteolytically digested proteins from isolated vesicles have confirmed previous results identifying the association of specific ion transporters with SVs. Further results indicate specific kinase association with SVs with measurable activity and, using immunoprecipitation and mass spectrometric methods, we are investigating endogenous kinase substrates. 2D chromatographic techniques (e.g., size exclusion followed by reversed-phase) have also been employed. Many expected proteins (e.g., synapsins, synuclein, syntaxin, and others) and peptides (e.g., tachykinins) have been identified. We also have obtained information and structural analysis on post-translational modifications, including glycosylation and acylation, on proteins and peptides from SVs and DCVs. Selective precipitation of proteins has allowed for detection of small proteins and peptides in much greater detail and has increased the number of neuropeptide assignments. Finally, we are investigating the gangliosides associated with CSVs and DCVs. A detailed study should provide not only valuable proteomic and biochemical information but also foster new method development in the examination of the types of molecules associated with vesicles.
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