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

中文翻译：

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突变热蛋白质组分析，用于表征蛋白质组内错义蛋白质变体及其相关表型。


温度敏感（TS）错义突变体已成为表征基本基因功能的基础。然而，缺乏一种公正的方法来分析 TS 错义突变体在功能蛋白质组背景下的生化和生物物理变化。我们应用基于 MS 的热蛋白质组分析 (TPP) 来研究错义突变对整个蛋白质组的影响，我们将其称为突变热蛋白质组分析 (mTPP)。这项研究描述了温度敏感性对 26S 蛋白酶体两个不同亚基中诱导错义突变的整体影响。 RNA-Seq 和全局蛋白质组学鉴定出的大多数改变在突变体之间是相似的，这可能表明两种错义变体中都发生了类似的功能破坏。然而，mTPP 的结果为每个突变体中 TS 表型的机制提供了独特的见解，揭示了仅使用稳态转录组和蛋白质组分析无法获得的独特变化。在计算上，多位点 λ 动力学模拟为 mTPP 实验结果提供了明确的支持。这项工作表明，mTPP 是一种精确的方法来测量含有错义突变体的蛋白质组的变化，而不需要大量的起始材料、针对感兴趣蛋白质的特异性抗体和/或生物系统的遗传操作。尽管实验是在允许的条件下进行的，但 mTPP 提供了对潜在蛋白质稳定性变化的见解，这些变化导致在非允许温度下观察到的戏剧性细胞表型。 总体而言，mTPP 以快速、无偏见的方式为错义突变功能障碍以及基因型与表型的联系提供了独特的机制见解。