Proteomics for Testing Hypotheses about Down Syndrome

用于检验唐氏综合症假设的蛋白质组学

• 批准号: 7082872
• 负责人: DAVID PATTERSON
• 金额: $ 27.65万
• 依托单位: UNIVERSITY OF DENVER (COLORADO SEMINARY)
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-25 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082872
• 关键词: Downs syndrome; cerebellum; free radical oxygen; gene expression; hippocampus; laboratory mouse; liquid chromatography mass spectrometry; mass spectrometry; matrix assisted laser desorption ionization; mitochondria; posttranslational modifications; protein quantitation /detection; proteolysis; proteomics; stable isotope; trisomy; two dimensional gel electrophoresis; western blottings

DESCRIPTION (provided by applicant): The overall goal of this proposal is to assess protein expression in brain regions from Ts65Dn mice [an animal model of Down syndrome (DS] using two dimensional gel electrophoresis/mass spectrometry (2DGE-MS). 2DgE-MS is necessary because altered mRNA expression does not always correlate with altered protein levels. A single gene frequently gives rise to multiple protein isoforms by posttranscriptional mechanisms, such as alternative splicing or posttranslational modifications. This effort will result in improved analytical methods, specifically 2DGE-MS quantitation and enhancement of the range of proteins analyzed by this approach. The method of choice will be difference gel electrophoresis (DGE) followed by MS analysis of the separated proteins. For proteins that are difficult to separate by 2DGE-MS, complementary approaches include fractionation on 1-dimensional SDS gels, slicing of the gels, proteolysis of the slices, and analysis by LC/MS/MS. For small proteins and peptides, identification will be done without proteolysis using known standards using our previously published procedures. We will compare the hippocampus and cerebellum of Ts65Dn mice and littermate normal controls at 3, 6 and 12 months of age because they span ages in which behavioral and histological changes occur in the Ts65Dn mice. We will initially focus on unfractionated extracts and mitochondria. Unfractionated extracts will be examined because of the richness of protein representation. Mitochondria will be analyzed because the mitochondrial proteome is relatively small, and because altered mitochondrial metabolism, including generation of reactive oxygen species, is hypothesized to be abnormal in DS. We hypothesize that: 1) the protein levels of many proteins, encoded not only on chromosome 21 (mouse chromosome 16) but throughout the genome, will be altered in the Ts65Dn mouse model of DS and therefore most probably in DS as well; 2) levels of proteins in the same biological (e.g., biochemical, signaling, developmental) pathway are coordinately regulated whether or not their genes are trisomic, 3) compensation for trisomy can occur via reduction of the levels of the proteins(s) encoded by the trisomic gene(s) or via reduction of the levels of other proteins in the biological pathway; 4) 2DGE-MS can identify coordinately regulated proteins, resulting in the identification of new biological pathways important for the phenotype of DS; 5) alternative processing of mRNAs commonly results in many protein isoforms from the same gene, greatly enhancing the complexity of the proteome; and 6) the mitochondrial proteome is altered in DS in ways that reflect altered mitochondrial metabolism in DS, contributing to the disabilities associated with DS. Understanding of the alterations in protein levels in the Ts65Dn mouse model of DS should lead to new approaches of ameliorate the cognitive and behavioral aspects of DS.

描述（由申请人提供）：该建议的总体目标是评估来自TS65DN小鼠的大脑区域的蛋白质表达[使用二维凝胶电泳/质谱分别（2DGE-MS）的唐氏综合症动物模型（DS]动物模型（DS）（2DGE-MS）（2DGE-MS）。2DGE-MS必须与单一的蛋白质相关，因为它与单个蛋白质相关。转录后的机制，例如替代剪接或翻译后的修饰。在一维SDS凝胶上分馏，切片，切片的蛋白水解以及LC/MS/MS分析。 对于小蛋白质和肽，使用我们先前发表的程序使用已知标准进行识别，而无需蛋白水解。 我们将比较3、6和12个月大的TS65DN小鼠和同窝型正常对照的海马和小脑，因为它们跨越了TS65DN小鼠的行为和组织学变化的年龄。 我们最初将专注于未分流的提取物和线粒体。由于蛋白质表示的丰富性，将检查未分流的提取物。 线粒体将进行分析，因为线粒体蛋白质组相对较小，并且由于线粒体代谢的改变（包括产生活性氧的产生）在DS中被认为是异常的。 我们假设：1）许多蛋白质的蛋白质水平不仅在21号染色体上编码（小鼠染色体16），而且在整个基因组中，都会在DS的TS65DN小鼠模型中改变，因此最可能在DS中。 2）在同一生物学（例如，生化，信号传导，发育）途径中的蛋白质水平是协同调节的，其基因是否是三异构体，3）三体性补偿可以通过降低蛋白质的水平来进行三叶素基因的蛋白质水平，而不是由三体基因基因或通过还原蛋白质的其他蛋白质的级别来实现。 4）2DGE-MS可以鉴定协调调节的蛋白质，从而鉴定出对DS表型很重要的新生物学途径。 5）mRNA的替代加工通常会导致许多来自同一基因的蛋白质同工型，从而大大提高了蛋白质组的复杂性。 6）线粒体蛋白质组在DS中以反映DS中线粒体代谢改变的方式改变，导致与DS相关的障碍。 了解DS的TS65DN小鼠模型中蛋白质水平的变化应导致改善DS的认知和行为方面的新方法。