Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome

Abstract

Temperature sensitive (TS) missense mutants have been foundational for characterization of essentialgene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TSmissense mutants within the context of their functional proteomes is lacking. We applied massspectrometry (MS) based thermal proteome profiling (TPP) to investigate the proteome-wide effects ofmissense mutations in an application that we refer to as mutant Thermal Proteome Profiling (mTPP).This study characterized global impacts of temperature sensitivity-inducing missense mutations in twodifferent subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and globalproteomics were similar between the mutants, which could suggest that a similar functional disruption isoccurring in both missense variants. Results from mTPP, however, provide unique insights into themechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were notobtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is aprecise approach to measure changes in missense mutant containing proteomes without the requirementfor large amounts of starting material, specific antibodies against proteins of interest, and/or geneticmanipulation of the biological system. Although experiments were performed under permissiveconditions, mTPP provided insights into the underlying protein stability changes that cause dramaticcellular phenotypes observed at non-permissive temperatures. Overall, mTPP provides uniquemechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in arapid, non-biased fashion.

Thermal proteome profiling reveals distinct target selectivity for differentially oxidized oxysterols

“…To define which shifts in melting temperatures were significant, two standard deviations from the median of all the calculated shifts was deemed appropriate, in line with previous reports. [17] In the screening process, proteins with a significant change in melting temperature following oxysterol exposure were filtered according their melting curves normalized to the lowest temperature. Proteins displaying a shift in the same direction (positive or negative ΔT m ) in all three replicates and with a curve plateau corresponding to a fraction of soluble protein less or equal to 0.5 were selected as potential targets (Figure 1A and 1C).…”

NF-κB Signaling Is Regulated by Fucosylation in Metastatic Breast Cancer Cells

“…Sample preparation, mass spectrometry analysis, bioinformatics, and data evaluation were performed in collaboration with the Proteomics Core Facility at the Indiana University School of Medicine (IUSM) [ 22 , 23 , 24 , 25 ]. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PXD021413 and 10.6019/PXD021413 [ 26 ].…”

Ref-1 Redox Activity Alters Cancer Cell Metabolism in Pancreatic Cancer: Exploiting This Novel Finding as a Potential Target  

“…Detailed method can be found in supplementary information and was adapted from literature reports 47,48 and vendor provided protocols. Brie y, cells were lysed using urea lysis buffer.…”