PROTEINS & PEPTIDES ASSOCIATED W/ NEURONAL DENSE CORE VESICLES

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。我们对两种类型的突触囊泡（SV）感兴趣，经典突触囊泡（CSV）和致密核囊泡（DCV）。我们实验室和其他实验室的研究表明，csv和dcv与神经生理学和神经病理生理学相关的特定蛋白质和神经化学共定位。我们的研究利用质谱和细胞生物学的工具，使用现有的方法，如毛细管高效液相色谱法，离子交换色谱法，串联质谱法和免疫组织化学，对SVs的成分进行了详细的蛋白质组学研究。利用分离囊泡中凝胶内蛋白水解消化蛋白的串联质谱数据的早期结果证实了先前的结果，即特异性离子转运蛋白与SVs之间存在关联。进一步的结果表明，特异性激酶与SVs的关联具有可测量的活性，并且使用免疫沉淀和质谱方法，我们正在研究内源性激酶底物。也采用了二维色谱技术（例如，粒径排除后反相）。许多预期的蛋白质（如突触蛋白、突触核蛋白、syntaxin等）和多肽（如速激肽）已被确定。我们还获得了来自SVs和dcv的蛋白质和肽的翻译后修饰的信息和结构分析，包括糖基化和酰化。蛋白质的选择性沉淀使得小蛋白质和多肽的检测更加详细，并增加了神经肽分配的数量。最后，我们正在研究与csv和dcv相关的神经节苷类。详细的研究不仅可以提供有价值的蛋白质组学和生化信息，还可以促进研究与囊泡相关的分子类型的新方法的发展。

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.