Proteomic Tools for the Comprehensive Analysis of Dopamine Transporter Topology

描述(申请人提供)：这份R21申请书(最多10页)是为了响应PAR-03-017“尖端基础研究奖(CEBRA)”而提交的，该奖项为高度创新或概念创新的研究提供支持，以促进对药物滥用和成瘾的了解。 膜蛋白脂质包埋残基的准确评估对于膜蛋白功能的预测和结构模型的建立至关重要。目前，膜蛋白拓扑预测算法最多只能预测+/-5~10个氨基酸。我们建议开发一种鸟枪式蛋白质组学方法，用于对复杂生物样本中完整的膜蛋白的脂质嵌入残基进行全局分析。我们将把这些方法应用于：1)一种直接的方法来表征人类多巴胺转运蛋白(HDAT)、运输HDAT的突变体和天然的DAT在大鼠脑组织中的拓扑结构；2)一种非定向的全局方法来表征从细胞和组织匀浆中富含的膜蛋白的拓扑结构。

DESCRIPTION (provided by applicant): This R21 application (10 pages max) is being submitted in response to PAR-03-017 "Cutting-Edge Basic Research Awards (CEBRA)" which provides support for highly innovative or conceptually creative research to advance the understanding of drug abuse and addiction. Accurate assessments of the lipid embedded residues of membrane proteins are crucial for the prediction of membrane protein function and the generation of structural models. Currently, membrane protein topology prediction algorithms are accurate to +/- 5 to 10 amino acids at best. We propose to develop a shotgun proteomic approach for the global analysis of lipid embedded residues of integral membrane proteins within a complex biological sample. We will apply these methods to: 1) a directed approach to characterize the topology of the human dopamine transporter (hDAT), trafficking mutants of hDAT, and native DAT in rat brain tissue and 2) an undirected global approach to characterize the topology of membrane proteins enriched from cell and tissue homogenates.