PROTEOMIC CHARACTERIZATION OF AGING IN CAENORHABDITIS ELEGANS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 老龄化过程并不像我们过去认为的那样是一个一成不变的现象，而是一个高度规范的过程。利用简单的模式生物，如蛔虫，秀丽线虫和果蝇，黑腹果蝇，在衰老研究方面取得了重大进展。到目前为止，蛋白质组中与年龄相关的修饰仅仅是用低分辨率双向电泳法概括地描述的，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，我们将研究线虫蛋白质组在正常老化过程中的变化。与加州大学旧金山分校的质谱学设施合作，我们将使用一种无凝胶的方法，将液相色谱(LC)质谱学和基于标签的定量相结合，以识别和定量年轻和老年线虫蛋白质提取物之间的差异。为了进行量化，我们将利用iTRAQ标记技术。该方法由4种等压标记试剂组成，可同时定量4种不同的样品。在胰酶消化后，老虫和幼虫的提取物都将被标记iTRAQ并结合。我们将进一步降低样品的复杂性，通过强阳离子交换层析分离多肽。我们将用纳米LC-电喷雾电离-四极飞行时间质谱仪(Nano-LC-ESI-QQ-TOF MS)对得到的组分进行分析。这种蛋白质组学对衰老的概述应该为整个衰老领域创建一个有价值的数据库。我们希望鉴定出一组随着年龄增长而改变的功能相关蛋白质，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，我们将对这些系统中有趣的组件进行上下调节，以衡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.