Carrasco del Amor, A. | bioRxiv Science

bioRxiv2022

Proteome integral solubility alteration assay combined with multi-criteria decision-making analysis for developing adverse outcome pathways

Understanding the biological impact of chemicals is hindered by the high number and diversity of compounds in the market. To simplify the chemical risk assessment, the adverse outcome pathway (AOP) method has arisen as a framework to predict the impact of chemical exposure on human and environmental health. The development of this predictive tool requires knowledge of the molecular interaction between chemicals and protein targets. Those molecular initiating events connect alterations of cellular function with physiological impairment. This strategy aims to focus on the complex biological interaction to predict the impact on health. The high-throughput identification of all chemical targets can be obtained by a proteomics-based thermal shift assay, however, selecting the priority target candidate is a biased process strongly dependent on expert knowledge and literature. Here, we unravel new molecular initiating event from a tested chemical combining the target deconvolution by the proteome integral solubility alteration (PISA) assay, and the target selection by an analytical hierarchy process (AHP) approach. In the proof-of-concept study, we identified by PISA assay 8 protein targets for 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) from the soluble proteome from hepatic cells containing 2824 proteins. The definition of the AHP approach facilitates the selection of heat shock protein beta-1 (Hspb1) as the most suitable protein for developing AOPs. Our results demonstrated that the process of target identification is independent from a chemical characterization, and that the process of data curation and target selection is less sensitive to lack of toxicological information. We anticipate that this innovative integration of methods could decipher the chemical-protein interactions from new chemicals including the new alternative chemicals designed for chemical replacement and that would discover new molecular initiating events to support more sustainable methodologies to gain time and resources in chemicals assessment. SYNOPSISOur combined methodologies can determine the most suitable target to develop adverse outcome pathways from the proteome-wide protein target identification.

pharmacology and toxicology↗

bioRxiv2022

Nonionic surfactants can modify the thermal stability of globular and membrane proteins interfering with the thermal proteome profiling principles to identify protein targets

The membrane proteins are essential targets to understand 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 was aiming to simultaneous 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 proteins solubility analyzed after the thermal shift assay were affected by the thermal 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 explore thermal-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.

biophysics↗