Nonionic Surfactants can Modify the Thermal Stability of Globular and Membrane Proteins Interfering with the Thermal Proteome Profiling Principles to Identify Protein Targets

Abstract

The membrane proteins are essential targets for understanding cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system-level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy aimed to simultaneously identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering protein solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed, globular, and membrane proteome. In the presence of surfactant micelles, the changes in protein solubility analyzed after the thermal shift assay was affected by the thermally dependent modification of the micellar size and its interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to exploring thermally independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanisms of action from pharma, biomedicine, analytical chemistry, or toxicology, and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.

The Introduction of Detergents in Thermal Proteome Profiling Requires Lowering the Applied Temperatures for Efficient Target Protein Identification

“…The introduction of non-ionic detergent NP40 to TPP has been demonstrated to be able to alter protein thermal stability in previous studies [ 18 , 21 ]. However, there is no definitive conclusion on whether the introduction of detergents impacts the target identification performance of TPP.…”

“…Reassuringly, the number of identified kinase targets was remarkably improved, and was comparable to that identified without detergents at 52 °C. A very recent study [ 21 ] also reported that the non-ionic detergent NP40 can modify protein thermal stability, and they therefore concluded that the introduction of detergents may not be compatible with the principles of TPP for target identification. Different from that conclusion, our results underscore the importance of using a lower temperature range in TPP for efficient target identification when utilizing detergents (either non-ionic or zwitterionic).…”

Stability-based approaches in chemoproteomics

“…Although this increases proteomic coverage (>8000 protein groups), recent research shows data indicating their destabilizing effects on the proteome. The average melting temperature across the proteome decreases upon the addition of detergent, leading to increased protein precipitation (Refs 48 , 49 ).…”

A Shift in Thinking: Cellular Thermal Shift Assay-Enabled Drug Discovery

“…In a recent publication by Kalxdorf et al, selective enrichment of glycosylated cell surface proteins was combined with CETSA MS to effectively identify compound binding to extracellular membrane receptors. Another approach is the use of detergents to solubilize the membrane-bound proteins and thereby increase the number of membrane proteins detected. , However, the use of a detergent remains challenging and interferes with the protein stability after the heat step . One successful example of the use of detergent in CETSA MS to identify membrane protein targets was reported by Carnero Corrales et al In this target deconvolution study of the autophagy inhibitor indophagolin, the purinergic receptor P2X4 was identified as a protein target in cell lysate with detergent.…”

“…3,27 However, the use of a detergent remains challenging and interferes with the protein stability after the heat step. 27 One successful example of the use of detergent in CETSA MS to identify membrane protein targets was reported by Carnero Corrales et al 28 In this target deconvolution study of the autophagy inhibitor indophagolin, the purinergic receptor P2X4 was identified as a protein target in cell lysate with detergent. This was confirmed by Western blot-based CETSA.…”