A PROTEOMIC APPROACH TO THE INDENTIFICATION OF PROTEINS*

描述（由申请人提供） 神经管缺陷（NTD）是最常见的先天性缺陷之一， 流行率从每1 000名新生儿1例到每1 000名新生儿8例不等， 关于种族群体。 各族裔群体之间的流行率差异如下： 与其他数据一致，表明存在遗传因素 与NTD有关。 动物研究证实了这种联系 表明对发育毒物诱导的NTD的敏感性不同， 在不同的近交系小鼠中。 人类和动物研究都表明 NTD的遗传成分是复杂的，可能涉及多个 的位点 此外，各种药物、化学品和物理制剂也是 已知会增加动物和某些情况下人类NTD的发病率。 尽管NTD的流行及其对NTD婴儿的相关影响 尽管NTD的病因学仍然知之甚少， 在过去20年的共同努力下。 缺乏了解是由于，在 部分原因是NTD的复杂性，以及直到最近， 实验方法，允许全球分析基因和 在复杂疾病过程中的蛋白质表达，如NTD。 内 目前，我们正在使用DNA微阵列来评估全球 暴露于高温的胚胎中的基因表达模式。 通过使用 来自敏感或耐药小鼠品系的胚胎 我们希望能找出特定的基因 与高血压引起的神经管畸形有关 在这份补助中，我们假设， 蛋白质组学的最新发展，特别是蛋白质组学的定量分析， 使用同位素编码的亲和标签的复杂蛋白质混合物（ICATS分析） 蛋白质磷酸化的分析，将提供重要的， 令人兴奋的新方法来研究蛋白质在病因学中的作用， 高血压引起的露脑畸形 利用这些新的蛋白质组学技术， 建议使用对高血压诱导的 露脑畸形，以确定蛋白质及其相关途径， 在高温致畸过程中发生了改变 为了在 在发育毒理学探索性（R21）研究资助的背景下，我们设置了 具体目标1：比较蛋白质 SWV（敏感）和C57 BL/6小鼠胚胎中的表达谱 （抗性）小鼠品系暴露于高温（43摄氏度）后， 第8.5天。 这一比较将通过进行定量分析来完成。 使用同位素编码的亲和标记物分析复杂的胚胎蛋白质混合物 （ICAT）。 具体目标2：比较小鼠中的磷蛋白表达谱 SWV（敏感）和C57 BL/6（抗性）小鼠品系的胚胎， 在第8.5天暴露于高温（43 ℃）。 这种比较将是 使用新开发的系统方法来分析 复杂蛋白质混合物中的蛋白质磷酸化。

DESCRIPTION (provided by applicant) Neural tube defects (NTDs) are among the most common of congenital defects, with prevalence rates ranging from 1 per 1000 to 8 per 1000 births depending on ethnic group. The differential prevalence among ethnic groups is consistent with other date suggesting that there are genetic factors associated with NTDs. Such an association is confirmed by animal studies showing that susceptibility to developmental toxicant-induced NTDs varies among different inbred strains of mice. Both human and animal studies suggest that the genetic component of NTDs is complex and likely to involve multiple loci. In addition, a variety of drugs, chemicals and physical agents are known to increase the incidence of NTDs in animals and in some cases humans. Despite the prevalence of NTDs and their associated toll on infants with NTDs and their families, the etiology of NTDs remains poorly understood despite concerted effort over the past 20 years. The lack of understanding is due, in part, to the complexity of NTDs and to the lack, until recently, of experimental approaches that allow a global analysis of patterns of gene and protein expression during complex disease processes like NTDs. Within currently funded grants, we are using DNA microarrays to assess global patterns of gene expression in embryos exposed to hyperthermia. By using embryos from strains of mice that are either sensitive or resistant to hyperthermia-induced exencephaly, we hope to identify specific genes associated with hyperthermia-induced NTDs. In this grant, we hypothesize that recent developments in proteomics, specifically quantitative analysis of complex protein mixtures using isotope-coded affinity tags (ICATS analysis) and the analysis of protein phosphorylation, will provide significant and exciting new methods to study the role of protein in the etiology of hyperthermia-induced exencephaly. Using these new proteomics technologies, we propose to use mouse strains differentially sensitive to hyperthermia-induced exencephaly to identify proteins and their associated pathways that are modified during hyperthermia teratogenesis. To accomplish this within the context of a Developmental Toxicology Exploratory (R21) Research Grant, we set forth the following specific aims: Specific Aim 1: compare protein expression profiles in mouse embryos of the SWV (sensitive) and C57BL/6 (resistant) strains of mice after exposure to hyperthermia (43 degrees C) on day 8.5. This comparison will be accomplished by undertaking a quantitative analysis of complex embryo protein mixtures using isotope-coded affinity tags (ICAT). Specific Aim 2: Compare phosphoprotein expression profiles in mouse embryos of the SWV (sensitive) and C57BL/6 (resistant) strains of mice after exposure to hyperthermia (43 degrees C) on day 8.5. This comparison will be accomplished using a newly-developed systematic approach to the analysis of protein phosphorylation in complex protein mixtures.