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 characterizing the proteins associated with synaptic vesicles (SV), many of which are glycosylated multi-pass membrane proteins. The synaptic protein SV2 is one example of an abundant extensively modified SV protein. This protein has an uncertain function and has an immature and mature forms which vary greatly in MW. The mature form has been shown to contain N-linked glycosylation as well as glycosaminoglycan chains. The goal of this project was to develop methods for enrichment, detection, and characterization of this and similar proteins. Early efforts have focused on using affinity based approaches from both synaptosomes and purified SVs from whole brain. These studies are utilizing the MALDI-TOF MS to get intact mass information from both glycosylated and de-glycosylated SV2 as well as proteolytic digests. Future studies will focus on using methodology such as capillary HPLC, tandem mass spectrometry, and immunohistochemistry as well as optimizing enzymatic digestion protocols.
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