The goal of the Multiplexing Protein Analysis Core (MPAC) is to provide users with specialized tools to determine the dynamic regulation of the proteome. As in the previous cycle, our primary service includes the rigorous, sensitive and precise quantification of panels of proteins relevant to the basic biology of aging and age-related disease. For this cycle, we expand the capabilities of the core by adding stable isotope tracer experiments with deuterium oxide (D2O) to measure the turnover of proteins. Although core facilities that offer discovery-based proteomics are relatively common, only a few cores offer these targeted methods. Further, the Core offers these assays in panels that interrogate specific biochemical pathways important in aging and can design new assays and panels on request for any protein from any animal with a sequenced genome. In addition, the Core can use its targeted approaches for post-translational modifications such as phosphorylation. There are also relatively few laboratories with the expertise to measure protein turnover rates using stable isotopes. Measuring synthetic rates with tracers requires proper study design, mass spectrometry with appropriate sample preparation and analysis, and correct interpretation of data. The advantages of D2O for Core users are significant. Specifically, it is cheap, highly sensitive, flexible, biologically inert, lends itself to long-term labeling, and can be used to measure the synthesis of a variety of molecules. The combination of D2O labeling and targeted proteomics in one sample allows users to understand changes in the content of individual proteins, the turnover processes that drive the changes, and mechanisms such as cell proliferation and ribosomal biogenesis that contribute to these changes. Finally, the analyses provided by the core are made on frozen samples, facilitating ease of sample collection for outside users. The Core proposes two specific aims: 1) Develop and apply high throughput multiplexed protein quantification for panels of proteins, including post-translational modifications, in experimental systems used by Geroscience investigators, including mice, rats, fruit flies, C.elegans, and yeast, and 2) Use stable isotope labeling and analysis in combination with multiplexed protein quantification to measure turnover of individual proteins as well as processes that contribute to the regulation of protein abundance. To accomplish the aims, the MPA Core uses selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) in tandem mass spectrometry systems or high-resolution accurate mass (HRAM) selected ion monitoring (SIM) in an orbitrap mass spectrometry system, as well as GC-MS based analysis of supportive measurements. The ability to adapt these procedures to multiple cell types, tissues, and model organisms make the MPA Core a significant resource for the aging research community.
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