Direct and Quantitative Proteomic Approaches in Xenopus

非洲爪蟾的直接定量蛋白质组学方法

• 批准号: 8710298
• 负责人: Frank Leo Conlon
• 金额: $ 18.97万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710298
• 关键词: Acute Lymphocytic Leukemia; Acute Myelocytic Leukemia; Address; Adult; Affect; Affinity; Amino Acid Sequence; Animals; Antibodies; Biological Models; Biotin; Boxing; Buffers; Castor; Collaborations; Communities; Complex; Core Facility; Cost Savings; DNA; Databases; Deposition; Development; Disease; Embryo; Enzymes; Feasibility Studies; Future; Generations; Genes; Genome; Goals; Heart; Holt Oram syndrome; Hypertension; Immunoprecipitation; Juvenile Myelomonocytic Leukemia; Label; Limb Development; Limb structure; Link; Liquid Chromatography; Mass Spectrum Analysis; Messenger RNA; Methodology; Molecular; Mutate; Mutation; Noonan Syndrome; PTPN11 gene; Patients; Peptide Sequence Determination; Peptides; Post-Translational Protein Processing; Procedures; Protein Databases; Protein phosphatase; Proteins; Proteomics; Protocols documentation; Reagent; Resources; Role; Series; Specificity; Staging; Streptavidin; Syndrome; System; Tadpoles; Technology; Testing; Time; Tissues; Transgenic Animals; Transgenic Organisms; Xenopus; Xenopus Proteins; Xenopus laevis; Zinc Fingers; base; cardiogenesis; chromatin immunoprecipitation; congenital heart disorder; cost; egg; embryo tissue; genome wide association study; human disease; in vivo; novel; protein complex; protein purification; public health relevance; receptor; research study; sperm cell; tandem mass spectrometry; transcription factor

DESCRIPTION (provided by applicant): ABSTRACT The goal of this proposal is to develop and optimize the technologies and reagents for use of directed and quantitative proteomic approaches in Xenopus. Specifically, we propose a series of feasibility studies based on three proteins, the T-box transcription factors TBX5, the zinc finger transcription factor CASZ1, and the non-receptor protein phosphatase SHP2. We have focused on these proteins based on their evolutionary conservation in sequence, expression, and function, their cellular localization, and the observation that these proteins are causative, or have association with human disease states: patients with Holt-Oram syndrome (HOS), a disease which also affects the heart and limbs, is associated with mis-sense mutations in Tbx5, genome wide association studies have implied a role for Casz1 in hypertension and high blood pressure, and patients with Noonan syndrome, a disease that also affects heart and limb development, frequently is associated with mis-sense mutations in Shp-2. Despite the crucial role for TBX5, CASZ1 and SHP-2 in development and human disease, the molecular mechanisms by which these proteins act in vivo remains to be established. To address these issues and to establish a set of general technologies for the use of directed proteomics approaches in Xenopus we propose to 1) establish a binary transgenic system in Xenopus for the isolation of protein and protein-DNA complexes and 2) establish the technologies, reagents and protocols for directed and quantitative proteomic approaches in Xenopus.

描述(由申请人提供)： 摘要这项建议的目标是开发和优化非洲爪哇定向和定量蛋白质组学方法的技术和试剂。具体地说，我们提出了一系列基于T-box转录因子TBX5、锌指转录因子CASZ1和非受体蛋白磷酸酶SHP2这三种蛋白质的可行性研究。我们关注这些蛋白质的基础是它们在序列、表达和功能上的进化保守性、它们的细胞定位，以及这些蛋白质是导致疾病或与人类疾病状态有关的观察结果：Holt-Oram综合征(HOS)(一种也会影响心脏和四肢的疾病)的患者与Tbx5的错义突变有关，全基因组研究表明CASZ1在高血压和高血压中起作用，而Noonan综合征(一种也影响心脏和肢体发育的疾病)的患者经常与SHP-2的错义突变有关。尽管TBX5、CASZ1和SHP-2在发育和人类疾病中起着至关重要的作用，但这些蛋白在体内发挥作用的分子机制仍有待建立。为了解决这些问题，并建立一套用于非洲爪哇定向蛋白质组学方法的通用技术，我们建议1)在非洲爪哇建立一个分离蛋白质和蛋白质-DNA复合体的二元转基因系统，以及2)建立非洲爪哇定向和定量蛋白质组学方法的技术、试剂和程序。