Multiplexing Protein Analysis Core

University Of Oklahoma Hlth Sciences Ctr, Oklahoma City OK

Investigators

Linked publications, trials & patents

Abstract

PROJECT SUMMARY – MULTIPLEXING PROTEIN ANALYSIS CORE (MPAC) The goal of the Multiplexing Protein Analysis Core (MPAC) is to provide users with specialized tools to determine the dynamic regulation of the proteome (i.e., proteostasis). As in the previous cycles, 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. The MPAC offers these assays as panels that interrogate specific biochemical pathways, can design new assays and panels on request for any protein from any animal with a sequenced genome, and can use its targeted approaches for post-translational modifications such as phosphorylation. Although core facilities that offer discovery-based proteomics are relatively common, only a few cores offer these targeted methods. For the current renewal, we add data-independent analysis (DIA) to greatly expand the depth and coverage of our quantitative analyses. We also provide stable isotope tracer experiments with deuterium oxide (D2O) as a service to measure the turnover of proteins and nucleic acids. These assays are design to further understand proteostatic regulation. There are 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 for a variety of other measurements when on board. 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 MPAC are made on frozen samples, facilitating ease of sample collection for outside users. The MPAC proposes two specific aims: 1) Develop and apply new high throughput multiplexed protein panels for quantification of multiple pathways important for geroscience investigators, and 2) Provide guidance and analysis of deuterium oxide (D2O) stable isotope labeling in combination with quantitative proteomic analysis and incorporation into nucleic acids (RNA and DNA) to determine synthesis rates of individual proteins as well as proteostatic mechanisms. To accomplish the aims, the MPAC uses selected reaction monitoring (SRM) with a triple quadrupole system and parallel reaction monitoring (PRM) and DIA analysis 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 MPAC a significant resource for the aging research community.