PROTEOMIC CHARACTERIZATION OF AGING IN CAENORHABDITIS ELEGANS

秀丽隐杆线虫衰老的蛋白质组学特征

• 批准号: 8363845
• 负责人: CYNTHIA J. KENYON
• 金额: $ 0.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363845
• 关键词: Affect; Aging; Aging-Related Process; Animal Model; Caenorhabditis elegans; Cations; Chromatography; Collaborations; Databases; Digestion; Drosophila genus; Drosophila melanogaster; Electrophoresis; Electrospray Ionization; Funding; Gel; Grant; Label; Liquid Chromatography; Longevity; Mass Spectrum Analysis; Measures; Mediating; Methods; Modification; National Center for Research Resources; Nematoda; Peptides; Principal Investigator; Process; Proteins; Proteome; Proteomics; Reagent; Research; Research Infrastructure; Resolution; Resources; Sampling; Source; System; Technology; Time; Trypsin; United States National Institutes of Health; age related; base; cost; interest; liquid chromatography mass spectrometry; mass spectrometer; multicatalytic endopeptidase complex; nano; normal aging; response; two-dimensional

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The aging process is not an immutable phenomenon as we used to believe, but a highly regulated process. Major advances in research on aging have been achieved using simple model organisms such as the roundworm, Caenorhabditis elegans and the fruit fly, Drosophila melanogaster. Until now age-related modifications in the proteome have been only summarily characterized with low-resolution two-dimensional electrophoresis, which precluded the identification of all but the most abundant proteins. In this project we will ask how the C. elegans proteome changes with normal aging. In collaboration with the UCSF Mass Spectrometry Facility, we will use a gel-free method by combining liquid chromatography (LC) mass spectrometry and a tag-based quantification to identify and quantify differences between protein extracts from young and old C. elegans. To perform the quantification we will take advantage of the iTRAQ labeling technology. This method consists of 4 isobaric tagging reagents allowing the quantification of four different samples at the same time. After trypsin digestion both old and young worm extracts will be labeled with iTRAQ and combined. We will further reduce the sample complexity by separating the peptides with strong cation exchange chromatography. We will analyze the fractions obtained with the nano-LC-electrospray ionization-quadrupole-time of flight mass spectrometer (nano-LC-ESI-Qq-TOF MS). This proteomics overview of aging should create a valuable database for the whole aging field. We expect to identify groups of functionally related proteins that are modified with aging, for example, proteins involved in the proteasome-mediated degradation or the unfolded-protein response. Some of the changes we see may cause, rather than reflect, aging. Therefore we will up- and down-regulate interesting components of these systems to measure how they affect lifespan.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 衰老过程并不像我们过去认为的那样是一种不可改变的现象，而是一个受到高度调控的过程。衰老研究的重大进展是利用简单的模型生物（例如蛔虫、秀丽隐杆线虫和果蝇、果蝇）取得的。到目前为止，蛋白质组中与年龄相关的修饰仅通过低分辨率二维电泳进行了概括性表征，这排除了除了最丰富的蛋白质之外的所有蛋白质的鉴定。在这个项目中，我们将询问秀丽隐杆线虫蛋白质组如何随着正常衰老而变化。与加州大学旧金山分校质谱设施合作，我们将使用无凝胶方法，结合液相色谱 (LC) 质谱和基于标签的定量来识别和量化年轻和年老秀丽隐杆线虫蛋白质提取物之间的差异。为了进行定量，我们将利用 iTRAQ 标记技术。该方法由 4 种同量异位标记试剂组成，可同时对四种不同的样品进行定量。胰蛋白酶消化后，老蠕虫和幼蠕虫提取物将用 iTRAQ 标记并合并。我们将通过强阳离子交换色谱分离肽，进一步降低样品的复杂性。我们将分析使用纳米 LC 电喷雾电离四极杆飞行时间质谱仪 (nano-LC-ESI-Qq-TOF MS) 获得的馏分。 衰老的蛋白质组学概述应该为整个衰老领域创建一个有价值的数据库。我们期望识别出随着衰老而改变的功能相关蛋白质组，例如参与蛋白酶体介导的降解或未折叠蛋白质反应的蛋白质。我们看到的一些变化可能会导致而不是反映衰老。因此，我们将上调和下调这些系统中有趣的组件，以衡量它们如何影响寿命。