Obtaining Increased Functional Proteomics Insights from Thermal Proteome Profiling through Optimized Melt Shift Calculation and Statistical Analysis

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 advancements and improvements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is even more apparent as TPP findings have reported more subtle melt shift changes related to protein post-translational 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. To benchmark InflectSSP, we have reanalyzed two publicly available datasets to demonstrate the performance of this publicly available R based program for TPP data analysis. Additionally, we report new findings following temporal treatment of human cells with the small molecule Thapsigargin which induces the unfolded protein response (UPR). InflectSSP analysis of our UPR study revealed highly reproducible target engagement over time while simultaneously providing new insights into the dynamics of UPR induction.

IB-DNQ and Rucaparib dual treatment alters cell cycle regulation and DNA repair in triple negative breast cancer cells

“…Analysis of TPP data with the R-package InflectSSP produces melt curves and calculates melt temperatures (Tm) and significant shifts in protein melt temperature. (45). TPP has been widely used to determine drug-target engagement (43), therefore we can apply it to test and compare the proteome-wide impacts on protein thermal stability across the different drug treatments.…”

“…Proteome-wide changes to protein-protein interactions, post-translational modifications, folding, and other biophysical properties of proteins can be assessed using thermal proteome profiling (TPP) (39)(40)(41)(42)(43). TPP is used to determine thermal stability of each protein under different conditions, which are then analyzed computationally to identify proteins and mechanisms impacted by treatment (44,45). Analysis of TPP data with the R-package InflectSSP produces melt curves and calculates melt temperatures (Tm) and significant shifts in protein melt temperature.…”

Implementation of an enriched membrane protein carrier channel for enhanced detection of membrane proteins in mass spectrometry-based thermal stability assays

“…Reporter ion isotope interference was observed in empty TMT channels when using a meSIILCC at 10-fold the total protein of the lowest heated MS-TSA Sigmoidal curve fitting of normalized reporter ion intensities is a common approach to determine melting temperatures (Tm) for MS-TSA. 13,36,37 However, isotope impurities within isobaric labeling reagents can be problematic when employing SIILCC. [38][39][40][41] We assessed isotopic interference from meSIILCC by conducting triplicate MS-TSA experiments using only dimethyl sulfoxide (DMSO)-treated samples with and without meSIILCC.…”

“…To a lesser extent, the 130C, 131N, 133N, and 134C channels also show much lower levels of isotope contamination, which could be addressed by the implementation of correction factors. 13,36…”