Proteomics for Testing Hypotheses about Down Syndrome

描述(由申请人提供)：这项建议的总体目标是使用双向凝胶电泳/质谱仪(2DGE-MS)评估Ts65Dn小鼠[唐氏综合症(DS)的动物模型]脑区域的蛋白质表达。2DGE-MS是必要的，因为改变的mRNA表达并不总是与改变的蛋白质水平相关。单个基因经常通过转录后机制产生多种蛋白质异构体，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定量和通过这种方法分析的蛋白质范围的增强。选择的方法是差示凝胶电泳法(DGE)，然后对分离的蛋白质进行MS分析。对于难以通过2DGE-MS分离的蛋白质，补充方法包括一维十二烷基硫酸钠凝胶分级、凝胶切片、切片蛋白质分解以及LC/MS/MS分析小蛋白质和多肽，将使用我们以前公布的程序在没有蛋白质分解的情况下使用已知标准进行鉴定。我们将比较Ts65Dn小鼠和3、6和12个月大的正常对照小鼠的海马体和小脑，因为它们跨越了Ts65Dn小鼠发生行为和组织学变化的年龄。我们首先将重点放在普通提取物和线粒体上。由于蛋白质的丰富性，将对未分离的提取物进行检查。将对线粒体进行分析，因为线粒体蛋白质组相对较小，而且假设DS患者线粒体代谢改变，包括产生活性氧物种，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(小鼠16号染色体)上编码，而且在整个基因组中都会在Ts65Dn小鼠模型中发生变化，因此很可能在DS中也会发生变化；2)同一生物途径(如生化、信号、发育)中的蛋白质水平受到协调调节，无论它们的基因是否为三体；3)对三体的补偿可以通过降低三体基因(S)编码的蛋白质(S)的水平或通过降低生物途径中其他蛋白质的水平来实现；4)2DGE-MS可以识别协同调节的蛋白质，从而识别对DS表型重要的新的生物学途径；5)mRNAs的替代处理通常导致同一基因的许多蛋白质异构体，极大地增加了蛋白质组的复杂性；以及6)DS中线粒体蛋白质组的改变反映了DS中线粒体代谢的改变，导致了DS相关的残疾。了解Ts65Dn小鼠DS模型中蛋白质水平的变化应该会导致改善DS认知和行为方面的新方法。

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.