Forward and reverse genetics approaches have identified many genes associated with organismal aging that are conserved across species. However, relatively little is known about how the proteome changes during aging in any organism. Using quantitative mass spectrometry together with protein and peptide-level fractionation, we can now report a major increase in coverage of the C. elegans proteome, identifying over 9,300 proteins in three biological replicates. The resulting data are being assembled into a searchable, online resource with a user-friendly graphical interface to provide convenient and open access to the community (https://www.peptracker.com/epd/). By quantifying these proteins in fertile, post-reproductive and aging animals, we show that there is a striking change in abundance of ~7% of the proteome during the course of aging. Our data reveal a concerted down-regulation of proteins involved in fatty acid, amino acid and carbohydrate metabolism, as well as peroxisome function, with increasing age. Interestingly, levels of PRX-5, which is responsible for the import of peroxisomal enzymes, also decrease during aging. Using a GFP reporter coupled to a peroxisomal targeting signal (PTS1), we observed that GFP-PTS1 is correctly localized to the peroxisome in young animals, whereas older animals showed GFP-PTS1 distributed across the cytosol and peroxisomes. This finding supports a model in which decreased PRX-5 levels contribute to an age-dependent reduction of peroxisomal protein localization and function. In summary, we have established quantitative proteomics methods to provide a deep coverage of the C. elegans proteome, with which we shed light on the importance of PRX-5 in maintaining peroxisome function during aging. Additionally, owing to innovations in our sample handling and processing methods, we are able to scale down the analysis of nematode proteomics to the level of a single animal. Thus, we can now reproducibly identify >3,000 proteins from a single nematode. This new single-worm proteomics pipeline opens up exciting new opportunities such as correlating protein variation with interesting phenotypic traits among individuals in a population. .
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