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无偏见的方法来检测和定量蛋白质和多肽，而不需要事先知道特定的蛋白质或多肽。然而，典型的蛋白质组学方案只检测给定组织中最丰富的蛋白质。虽然这些丰富的分子的变化对于理解药物滥用的后果很重要，但检查那些不那么丰富的分子也很重要，比如那些参与细胞间信号传输的分子。我们开发了一种从脂肪/脂肪小鼠脑中选择性地分离多肽加工中间体的新方法，并对该方法进行了改进，使其能够通过差异同位素标记来定量测定两组不同动物中多肽的相对水平。通过这种技术分离的多肽是羧基肽酶E的底物，羧基肽酶E是许多神经内分泌肽生物合成中的一种重要酶。 在目标1中，我们将使用我们的定量多肽组学方法来检测可卡因或甲基苯丙氨酸给药对特定脑区如海马体、纹状体、前额叶皮质和下丘脑的多肽相对水平的影响。 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多肽在野生型小鼠中是否也有类似的改变。由于肽组学技术由于其他分子的高背景而无法在野生型小鼠中检测到神经肽，这一目标将使用放射免疫分析和Northern印迹分析等传统技术来检测多肽和mRNA的水平。 综上所述，这些研究将更好地理解神经肽在药物滥用中的调节，无论是已知的还是新颖的。

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.