PROTEOMIC CHARACTERIZATION OF AGING IN CAENORHABDITIS ELEGANS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 衰老过程并不像我们过去认为的那样是一个不可改变的现象，而是一个高度调节的过程。衰老研究的主要进展是使用简单的模式生物，如蛔虫、秀丽隐杆线虫和果蝇、黑腹果蝇。到目前为止，蛋白质组中与年龄相关的修饰仅用低分辨率二维电泳进行概括性表征，这排除了除最丰富的蛋白质外的所有蛋白质的鉴定。在这个项目中，我们将问如何C。线虫蛋白质组随衰老而变化。与UCSF质谱设施合作，我们将使用无凝胶方法，通过结合液相色谱（LC）质谱和基于标签的定量来识别和定量年轻和老年C蛋白提取物之间的差异。优雅为了进行定量，我们将利用iTRAQ标记技术。该方法由4种同量异位素标记试剂组成，允许同时定量4种不同的样品。胰蛋白酶消化后，将用iTRAQ标记老龄和幼龄蠕虫提取物并合并。我们将通过强阳离子交换色谱分离肽来进一步降低样品的复杂性。我们将用nano-LC-电喷雾电离-四极杆-飞行时间质谱仪（nano-LC-ESI-Qq-TOF MS）分析获得的级分。 这种蛋白质组学的衰老概述应该为整个衰老领域创建一个有价值的数据库。我们希望能够确定功能相关的蛋白质组，这些蛋白质随年龄的变化而改变，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，老化。因此，我们将上调和下调这些系统的有趣组件，以测量它们如何影响寿命。

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.