CHARACTERIZATION OF THE SYNAPTIC PROTEIN SV2

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。神经递质的囊泡运输促进了突触传递。突触囊泡的性质和组成已被越来越多地研究。我们感兴趣的是表征与突触囊泡（SV）相关的蛋白质，其中许多是糖基化的多通道膜蛋白。突触蛋白SV2是一个丰富的广泛修饰的SV蛋白的例子。该蛋白具有不确定的功能，在MW中有不成熟和成熟的形式，差异很大。成熟的形式已被证明含有n链糖基化以及糖胺聚糖链。该项目的目标是开发这种蛋白质和类似蛋白质的富集、检测和表征方法。早期的研究主要集中在利用基于亲和性的方法从突触体和纯化的全脑SVs中进行研究。这些研究利用MALDI-TOF质谱从糖基化和去糖基化的SV2以及蛋白水解消化中获得完整的质量信息。未来的研究将集中在使用毛细管高效液相色谱、串联质谱、免疫组织化学等方法学以及优化酶消化方案上。

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.