Proteomics for Testing Hypotheses about Down Syndrome

描述（由申请人提供）：本提案的总体目标是使用二维凝胶电泳/质谱（2DGE-MS）评估Ts65Dn小鼠[唐氏综合征（DS）动物模型]脑区域的蛋白质表达。2DgE-MS是必要的，因为mRNA表达的改变并不总是与蛋白质水平的改变相关。单个基因经常通过转录后机制产生多种蛋白质亚型，如选择性剪接或翻译后修饰。这一努力将导致分析方法的改进，特别是2DGE-MS定量和通过该方法分析的蛋白质范围的增强。选择的方法是差异凝胶电泳（DGE），然后对分离的蛋白质进行质谱分析。对于难以通过2DGE-MS分离的蛋白质，补充方法包括在一维SDS凝胶上分离，凝胶切片，切片蛋白水解，LC/MS/MS分析。对于小蛋白和多肽，鉴定将在没有蛋白水解的情况下使用已知的标准，使用我们之前公布的程序。我们将比较Ts65Dn小鼠的海马和小脑在3、6和12个月大时与正常对照的窝鼠，因为它们跨越了Ts65Dn小鼠发生行为和组织学变化的年龄。我们将首先关注未分离提取物和线粒体。未分离的提取物将被检查，因为丰富的蛋白质表示。线粒体将被分析，因为线粒体蛋白质组相对较小，因为线粒体代谢的改变，包括活性氧的产生，被假设在DS中是异常的。我们假设：1)在Ts65Dn小鼠DS模型中，不仅在21号染色体（小鼠16号染色体）上编码，而且在整个基因组中编码的许多蛋白质的蛋白质水平将发生改变，因此很可能也发生在DS中；2)无论基因是否三体，同一生物（如生化、信号、发育）途径中的蛋白质水平都是协调调节的；3)三体补偿可以通过降低三体基因编码的蛋白质水平或降低生物途径中其他蛋白质的水平来实现；4) 2DGE-MS可以鉴定出协同调节的蛋白，从而鉴定出对DS表型有重要意义的新的生物学通路；5) mrna的替代加工通常会导致来自同一基因的许多蛋白质异构体，极大地提高了蛋白质组的复杂性；6)线粒体蛋白质组在退行性痴呆中以反映线粒体代谢改变的方式发生改变，从而导致退行性痴呆相关的残疾。了解Ts65Dn小鼠退行性椎体滑移模型中蛋白水平的变化，将为改善退行性椎体滑移的认知和行为方面带来新的方法。

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.