The ubiquitin-proteasome system (UPS) is a key protein regulator of proteins. However, a full compendium of substrates of the UPS does not exist. As a protein complex, the proteasome can serve as a model for the analysis of protein complex dynamics in the presence of protein sequence variants such as missense mutations. Analysis of the impact of missense mutations on protein stability is currently intractable at a high-throughput scale. Yeast strains containing missense mutations in proteasome subunits were used to analyze the effects of mutations on global protein abundance and stability via quantitative mass spectrometry. In the mutants analyzed to date, over 1,200 proteins increased in abundance over WT. Next, we developed a new application of the cellular thermal shift assay (CETSA) to profile the thermal impacts of mutations in a single protein on the full proteome. This approach enables analysis of the effects of changes in subunits of the proteasome on the proteasome as a complex. Addition of CETSA led to more specific protein changes in mutants relative to WT, with many of the changes being within the proteasome itself. Successful development of this method provides a high-throughput way to profile the global effects of missense mutations on protein function and to characterize resulting changes in PPI networks.
Sarah is a graduate student in the lab of Amber Mosley in the Department of Biochemistry and Molecular Biology at Indiana University School of Medicine. The Mosley lab utilizes both proteomic and genomic approaches to study the regulation of transcription by RNA polymerase II in Saccharomyces cerevisiae. Sarah is currently developing a quantitative proteomics approach that couples an adapted cellular thermal shift assay to high resolution mass spectrometry to profile the thermal stability of proteins with missense mutations. This application provides a high throughput method to characterize the impacts of disease-associated missense mutations in proteins on the global proteome. Sarah is a 2018 TMT Research Award recipient (Bronze level).