PROTEINS & PEPTIDES ASSOCIATED W/ NEURONAL DENSE CORE VESICLES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。神经递质的囊泡运输促进了突触的传递。突触囊泡的性质和组成已得到越来越多的研究。我们对两种类型的突触小泡(SV)感兴趣，即经典突触小泡(CSV)和致密核心小泡(DCV)。我们实验室和其他实验室的研究表明，CSV和DCV具有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。我们的研究利用了质谱学和细胞生物学的工具，利用现有的方法，如毛细管高效液相色谱、离子交换层析、串联质谱学和免疫组织化学等，对SVS的成分进行了详细的蛋白质组学研究。从分离的囊泡中提取的凝胶内蛋白水解性消化蛋白的串联质谱学数据的早期结果证实了以前的结果，即特定的离子转运体与SVS的关联。进一步的结果表明，与SVS相关的特定的激酶具有可测量的活性，并且，使用免疫沉淀和质谱学方法，我们正在研究内源性的激酶底物。还采用了二维色谱技术(例如，先进行尺寸排除，然后进行反相)。许多预期的蛋白质(如突触素、突触素、突触素等)和多肽(如速激肽)已被鉴定。我们还获得了SVS和DCVS的蛋白质和多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可以更详细地检测到小的蛋白质和多肽，并增加了神经肽的分配数量。最后，我们正在调查与CSV和DCV相关的神经节苷脂。一项详细的研究不仅应该提供有价值的蛋白质组和生化信息，而且还应该促进检查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.