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Proteomics for Testing Hypotheses about Down Syndrome

用于检验唐氏综合症假设的蛋白质组学

基本信息

批准号：
7248057
负责人：
DAVID PATTERSON
金额：
$ 26.85万
依托单位：
UNIVERSITY OF DENVER (COLORADO SEMINARY)
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-25 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7248057
关键词：
Age Age-Months Alternative Splicing Animal Model Behavioral Biochemical Biological Birth Brain Brain region Cerebellum Chromosomes, Human, Pair 16 Chromosomes, Human, Pair 21 Cognitive Compatible Condition Development Down Syndrome Exhibits Financial compensation Fractionation Gel Gene Proteins Generations Genes Genetic Genome Goals Hippocampus (Brain)Human Incidence Individual Isoelectric Focusing Laboratories Lead Live Birth Mass Spectrum Analysis Metabolic Metabolism Methods Mitochondria Mitochondrial Proteins Molecular Weight Mus Nervous System Physiology Neuraxis Pathway interactions Peptides Persons Phenotype Play Population Post-Translational Protein Processing Procedures Process Protein Analysis Protein Isoforms Proteins Proteolysis Proteome Proteomics Publishing Range Reactive Oxygen Species Role Sampling Set protein Signal Transduction Slice Standards of Weights and Measures System Testing Trisomy Two-Dimensional Gel Electrophoresis analytical method base comparative disability gel electrophoresis improved mRNA Expression mouse Ts65Dn mouse model novel strategies protein expression research study

项目摘要

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）：本提案的总体目标是使用二维凝胶电泳/质谱（2DGE-MS）评估Ts 65 Dn小鼠[唐氏综合征（DS）的动物模型]脑区域中的蛋白质表达。 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。单个基因通常通过转录后机制，如选择性剪接或翻译后修饰，产生多种蛋白质亚型。这一努力将导致改进的分析方法，特别是2DGE-MS定量和增强的范围内的蛋白质分析的这种方法。选择的方法将是差异凝胶电泳（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，补充方法包括在一维SDS凝胶上进行分级分离、凝胶切片、切片的蛋白水解以及通过LC/MS/MS进行分析。对于小蛋白质和肽，将使用我们先前公布的程序使用已知标准品在没有蛋白水解的情况下进行鉴定。我们将比较海马和小脑的Ts 65 Dn小鼠和同窝正常对照组在3个月，6个月和12个月的年龄，因为他们跨越年龄的行为和组织学变化发生在Ts 65 Dn小鼠。我们将首先关注未分级提取物和线粒体。由于蛋白质表达的丰富性，将检查未分级提取物。将分析线粒体，因为线粒体蛋白质组相对较小，并且因为改变的线粒体代谢（包括活性氧的产生）假设在DS中是异常的。我们假设：1）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在DS的Ts 65 Dn小鼠模型中改变，因此最有可能在DS中也改变; 3）通过降低由三体基因编码的蛋白质的水平或通过降低生物学途径中的其它蛋白质的水平，可以发生对三体的补偿; 4）2DGE-MS可以识别协同调控的蛋白质，从而鉴定出对DS表型重要的新的生物学途径; 5）mRNA的替代加工通常导致来自相同基因的许多蛋白质同种型，大大提高了蛋白质组的复杂性;以及6）DS中线粒体蛋白质组以反映DS中线粒体代谢改变的方式改变，导致与DS相关的残疾。了解DS Ts 65 Dn小鼠模型中蛋白质水平的改变，应导致改善DS认知和行为方面的新方法。