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.
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