Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP
Year: 2023
Type: article
Abstract: Thermal proteome profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein-ligand, protein-protein, and protein-RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with new approaches leading to advancements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is important to fill as TPP findings have reported subtle melt shift changes related to signaling events such as protein posttranslational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. InflectSSP incorporates calculation of a "melt coefficient" into the pipeline with production of average melt curves for biological replicate studies to aid in identification of proteins with significant melts. To benchmark InflectSSP, we have reanalyzed two previously reported datasets to demonstrate the performance of our publicly available R-based program for TPP data analysis. We report new findings following temporal treatment of human cells with the small molecule thapsigargin that induces the unfolded protein response as a consequence of inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase 2A. InflectSSP analysis of our unfolded protein response study revealed highly reproducible and statistically significant target engagement over a time course of treatment while simultaneously providing new insights into the possible mechanisms of action of the small molecule thapsigargin.
Source: Molecular & Cellular Proteomics
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $2,800
Funders: Indiana University, Indiana Clinical and Translational Sciences Institute, Ralph W. and Grace M. Showalter Research Trust Fund, National Institutes of Health, National Institute on Aging, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Center for Advancing Translational Sciences
Awards: Regulation and Function of Integrated Stress Response, Indiana Medical Scientist/Engineer Training Program, Indiana Clinical and Translational Sciences Institute, Proteomic and functional analysis of missense variants of APOE associated with Alzheimer disease risk, Indiana Diabetes Research Center, Indiana Medical Scientist/Engineer Training Program, Thermal proteome profiling for analysis of protein sequence variants in human genetic disease, Indiana University Melvin and Bren Simon Comprehensive Cancer Center Support Grant
OpenAlex ID: W4385648907