A PROTEOMIC APPROACH TO THE INDENTIFICATION OF PROTEINS*

鉴定蛋白质的蛋白质组学方法*

• 批准号: 6501200
• 负责人: PHILIP E MIRKES
• 金额: $ 15.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6501200
• 关键词: DNA damage; congenital brain disorder; developmental neurobiology; disease /disorder etiology; disease /disorder proneness /risk; embryo /fetus drug adverse effect; embryo /fetus tissue /cell culture; environmental exposure; gene expression; laboratory mouse; microarray technology; neural plate /tube; neurogenetics; phosphoproteins; phosphorylation; protein protein interaction; protein structure function; proteomics; teratogens

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.

描述（由申请人提供） 神经管缺陷（NTD）是先天性缺陷中最常见的， 患病率从每1000到1000个出生的1000次出生率不等 在族裔群体上。 种族之间的差异患病率是 与其他日期一致，表明存在遗传因素 与NTD相关联。 动物研究证实了这种关联 表明对发育毒物诱导的NTD的敏感性有所不同 在不同的近交菌株中。 人类和动物研究都表明 NTD的遗传成分很复杂，并且可能涉及多个 基因座。 此外，多种药物，化学物质和物理剂是 已知会增加动物中NTD的发病率，在某些情况下是人类。 尽管NTD及其对NTD的婴儿的相关损失 和他们的家人，尽管 在过去的20年中，一致的努力。 缺乏理解是到期的 部分，直到最近，NTD的复杂性和缺乏 实验方法允许全球分析基因模式和 在复杂疾病过程中的蛋白质表达等NTD。 之内 当前资助的赠款，我们正在使用DNA微阵列评估全球 暴露于高温的胚胎中基因表达的模式。 通过使用 对小鼠菌株的胚胎，对敏感或抗性 热疗引起的外导体，我们希望识别特定的基因 与热疗诱导的NTD相关。 在这笔赠款中，我们假设 蛋白质组学的最新发展，特别的定量分析 使用同位素编码的亲和力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和 令人兴奋的新方法来研究蛋白质在病因中的作用 热疗诱导的外导体。 使用这些新的蛋白质组学技术，我们 建议使用对热疗诱导的小鼠菌株差异敏感的 除外，以鉴定蛋白质及其相关途径 在高温畸胎期内修饰。 在 发育毒理学探索性（R21）研究赠款的背景，我们设定 提出以下特定目的：特定目标1：比较蛋白质 SWV（敏感）和C57BL/6的小鼠胚胎中的表达曲线 暴露于高温（43度C）后的（抗性）小鼠菌株 第8.5天。 这种比较将通过进行定量来完成 使用同位素编码的亲和力标签分析复杂的胚胎蛋白混合物 （ICAT）。 特定目标2：比较小鼠中的磷蛋白表达谱 SWV（敏感）和C57BL/6（抗性）菌株的胚胎后的胚胎 第8.5天暴露于高温（43度C）。 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。