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

摘要: Temperature-sensitive (TS) missense mutants have been foundational for characterization of essential gene function. However, an unbiased approach analysis biochemical and biophysical changes in TS within the context their functional proteomes is lacking. We applied MS-based thermal proteome profiling (TPP) to investigate proteome-wide effects mutations application that we refer as mutant (mTPP). This study characterized global impacts temperature sensitivity–inducing two different subunits 26S proteasome. The majority alterations identified by RNA-Seq proteomics were similar between mutants, which could suggest a disruption occurring both variants. Results from mTPP, however, provide unique insights into mechanisms contribute phenotype each mutant, revealing distinct not obtained using only steady-state transcriptome analyses. Computationally, multisite λ-dynamics simulations add clear support mTPP experimental findings. work shows precise measure mutant–containing without requirement large amounts starting material, specific antibodies against proteins interest, and/or genetic manipulation biological system. Although experiments performed under permissive conditions, provided underlying protein stability cause dramatic cellular phenotypes observed at nonpermissive temperatures. Overall, provides mechanistic mutation dysfunction connection genotype rapid, nonbiased fashion.