Thermal proteome profiling: unbiased assessment of protein state through heat-induced stability changes

Abstract

In recent years, phenotypic-based screens have become increasingly popular in drug discovery. A major challenge of this approach is that it does not provide information about the mechanism of action of the hits. This has led to the development of multiple strategies for target deconvolution. Thermal proteome profiling (TPP) allows for an unbiased search of drug targets and can be applied in living cells without requiring compound labeling. TPP is based on the principle that proteins become more resistant to heat-induced unfolding when complexed with a ligand, e.g., the hit compound from a phenotypic screen. The melting proteome is also sensitive to other intracellular events, such as levels of metabolites, post-translational modifications and protein-protein interactions. In this review, we describe the principles of this approach, review the method and its developments, and discuss its current and future applications. While proteomics has generally focused on measuring relative protein concentrations, TPP provides a novel approach to gather complementary information on protein stability not present in expression datasets. Therefore, this strategy has great potential not only for drug discovery, but also for answering fundamental biological questions.

Thermal proteome profiling for interrogating protein interactions

“…More commonly, TPP experiments involve chemical [e.g., drug (Azimi et al, 2018;Becher et al, 2016;Hu et al, 2019;Huber et al, 2015;Kitagawa et al, 2017;Mateus et al, 2018Mateus et al, , 2016Reinhard et al, 2015;Savitski et al, 2014Savitski et al, , 2018 . Some of the perturbations can be applied in a dose-dependent manner (Becher et al, 2016) or time-dependent manner Dai et al, 2018) to improve data analysis or facilitate mechanistic understanding of the perturbation (Fig 2).…”

“…Some of the perturbations can be applied in a dose‐dependent manner (Becher et al , ) or time‐dependent manner (Becher et al , ; Dai et al , ) to improve data analysis or facilitate mechanistic understanding of the perturbation (Fig ). Using this approach, it has been possible to deconvolute drug targets (Savitski et al , , ; Huber et al , ; Reinhard et al , ; Becher et al , ; Mateus et al , , ; Kitagawa et al , ; Azimi et al , ; Hu et al , ) and enzyme substrates (preprint: Saei et al , ), study metabolic shifts (Becher et al , ; Dai et al , ; Mateus et al , ), or identify protein–protein interactions (Tan et al , ).…”

Thinking Outside the Box—Novel Antibacterials To Tackle the Resistance Crisis

“…[133] Thermal proteome profiling allows an unbiased search of protein targets in live cells using parent drugs without the need for modification. Although thermal proteome profiling has been widely applied in eukaryotic systems [136,137] there are only af ew examples of its use in microbes.H owever,i nitial studies show that this method can also be applied in live bacterial cells in atargeted way (through Western blot) [138,139] and in an unbiased way in bacterial lysate (by MS-based proteomics). [134,135] Protein stability is assessed by compoundinduced changes in the protein precipitation temperatures which can either be analyzed for selected proteins by Western Blot, or in an unbiased way using MS-based proteomics ( Figure 5b).…”

Über bisherige Denkweisen hinaus – neue Wirkstoffe zur Überwindung der Antibiotika‐Krise

“…Die Autoren quantifizierten etwa 750 Metaboliten nach Antibiotika-Exposition und konnten konzentrationsabhängige und stoffklassenspezifische ¾nderungen in verschiedenen Stoffwechselwegen beobachten. Während das thermische Proteom-Profiling bereits ausgiebig in eukaryotischen Systemen verwendet wurde, [136,137] gibt es bisher nur wenige Beispiele fürd ie Anwendung in Mikroorganismen. [133] Das thermische Proteom-Profiling ermçglicht es unter Verwendung der ursprünglichen, nichtmodifizierten Wirkstoffe,d ie jeweiligen Zielproteine in lebenden Zellen zu bestimmen.…”