GLYCOLIPID METABOLISM IN SINGLE CELLS (RMI)

描述(由申请人提供)：这份R21/R33提案结合了一个国际团队的努力，以开发工具和方法来监测单个哺乳动物细胞中神经节苷脂代谢组的子集。 在该项目为期两年的R21阶段，将使用化学和酶促方法合成一套8种荧光底物和标准。该集合包括沿两条不同糖脂代谢途径的所有已知代谢物。标准品和底物将用四种不同的荧光标记制备，这样就可以同时访问代谢途径中的不同点。NG108-15和/或PC12细胞将与底物孵育，底物将进行生物合成和生物降解。只有那些保留荧光标记的反应产物才会在我们的仪器中产生可检测到的信号，从而允许仔细剖析特定的代谢途径。将为该项目组装一个四光谱通道激光诱导荧光探测器。这个检测系统将允许对四组不同的代谢产物进行亚原子分子检测，这将为表征不同途径的代谢物提供一个特别强大的工具。 在项目为期一年的R33阶段，这套试剂和仪器将应用于单细胞新陈代谢的研究。 该项目的长期目标是监测单个神经元中完整的神经节苷脂代谢组。

DESCRIPTION (provided by applicant): This R21/R33 proposal combines the efforts of an international team to develop tools and methods to monitor a subset of the ganglioside metabolome in single mammalian cells. In the two-year duration R21 phase of the project, a set of eight fluorescent substrates and standards will be synthesized using chemical and enzymatic methods. The set consists of all known metabolites along two different glycolipid metabolic pathways. The standards and substrates will be prepared with four different fluorescent labels, so that different points in the metabolic pathway can be accessed simultaneously. NG108-15 and/or PC12 cells will be incubated with the substrates, which will undergo biosynthesis and biodegradation. Only those reaction products that preserve the fluorescent label will generate a detectable signal in our instrument, allowing careful dissection of specific metabolic pathways. A four-spectral channel laser-induced fluorescence detector will be assembled for this project. This detection system will allow the subattomole detection of four different sets of metabolic products, which will provide a particularly powerful tool for the characterization of metabolites along different pathways. During the one-year duration R33 phase of the project, this set of reagents and instrumentation will be applied to the study of metabolism in single cells. The long-term goal of this project is to monitor the complete ganglioside metabolome in single neurons.