Peptidomics of Cocaine and Amphetamine Abuse

描述（由申请人提供）： 蛋白质组学方法提供了一种检测和定量蛋白质和肽的非偏倚方式，而无需特定蛋白质或肽的先验知识。然而，典型的蛋白质组学方案仅检测给定组织中最丰富的蛋白质。虽然这些丰富分子的变化对于理解药物滥用的后果可能很重要，但检查不太丰富的分子也很重要，例如参与细胞之间信号传递的分子。我们已经开发了一种新的方法，选择性地分离肽加工中间体脂肪/脂肪小鼠脑，并修改了这种方法，允许定量的相对水平的肽在两个不同的动物组的差异同位素标记。通过这种技术分离的肽是羧肽酶E的底物，羧肽酶E是许多神经内分泌肽生物合成中的重要酶。 在目标1中，我们将使用我们的定量肽组学方法来检查可卡因或甲基苯丙胺给药对特定脑区（如海马、纹状体、前额叶皮质和下丘脑）中肽的相对水平的影响。 目标2将测试在肥胖小鼠中发现的被慢性可卡因或甲基苯丙胺改变的任何肽在野生型小鼠中是否也被类似地改变。由于肽组学技术由于来自其他分子的高背景而不能检测野生型小鼠中的神经肽，因此本目标将使用传统技术如放射免疫测定和北方印迹分析来检查肽和mRNA水平。 总之，这些研究将提供一个更好的了解的调节神经肽，无论是已知的和新的，在药物滥用。

DESCRIPTION (provided by applicant): Proteomics methods provide a non-biased way of detecting and quantitating proteins and peptides, without prior knowledge of the particular protein or peptide. However, typical proteomics schemes detect only the most abundant proteins in a given tissue. While changes in these abundant molecules can be important in understanding the consequence of drug abuse, it is also important to examine the less abundant molecules such as those involved in signal transmission between cells. We have developed a novel method to selectively isolate peptide processing intermediates from fat/fat mouse brain, and have modified this method to permit the quantitation of relative levels of the peptides in two different groups of animals by differential isotopic labeling. The peptides isolated by this technique are substrates of carboxypeptidase E, an important enzyme in the biosynthesis of numerous neuroendocrine peptides. In Aim 1, we will use our quantitative peptidomics method to examine the effect of cocaine or methamphetarnine administration on the relative levels of peptides in specific brain areas such as the hippocampus, striatum, prefrontal cortex, and hypothalamus. Aim 2 will test whether any peptides found to be altered by the chronic cocaine or methamphetamine in the fat mice are similarly altered in wild type mice. Because the peptidomics technique cannot detect neuropeptides in wild type mice due to the high background from other molecules, this Aim will use traditional techniques such as radioimmunoassay and Northern blot analysis to examine peptide and mRNA levels. Taken together, these studies will provide a better understanding of the regulation of neuropeptides, both known and novel, in drug abuse.