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|Title:||Cellular thermal shift assay for the identification of drug-target interactions in the Plasmodium falciparum proteome||Authors:||Dziekan, Jerzy Michal
Go, Ka Diam
Lim, Yan Ting
Wang, Loo Chien
Sobota, Radoslaw M.
|Keywords:||Science::Biological sciences||Issue Date:||2020||Source:||Dziekan, J. M., Wirjanata, G., Dai, L., Go, K. D., Yu, H., Lim, Y. T., Chen, L., Wang, L. C., Puspita, B., Prabhu, N., Sobota, R. M., Nordlund, P. & Bozdech, Z. (2020). Cellular thermal shift assay for the identification of drug-target interactions in the Plasmodium falciparum proteome. Nature Protocols, 15(6), 1881-1921. https://dx.doi.org/10.1038/s41596-020-0310-z||Project:||MOH/IAFCAT2/004/2015
|Journal:||Nature Protocols||Abstract:||Despite decades of research, little is known about the cellular targets and the mode of action of the vast majority of antimalarial drugs. We recently demonstrated that the cellular thermal shift assay (CETSA) protocol in its two variants: the melt curve and the isothermal dose-response, represents a comprehensive strategy for the identification of antimalarial drug targets. CETSA enables proteome-wide target screening for unmodified antimalarial compounds with undetermined mechanisms of action, providing quantitative evidence about direct drug-protein interactions. The experimental workflow involves treatment of P. falciparum-infected erythrocytes with a compound of interest, heat exposure to denature proteins, soluble protein isolation, enzymatic digestion, peptide labeling with tandem mass tags, offline fractionation, and liquid chromatography-tandem mass spectrometry analysis. Methodological optimizations necessary for the analysis of this intracellular parasite are discussed, including enrichment of parasitized cells and hemoglobin depletion strategies to overcome high hemoglobin abundance in the host red blood cells. We outline an effective data processing workflow using the mineCETSA R package, which enables prioritization of drug-target candidates for follow-up studies. The entire protocol can be completed within 2 weeks.||URI:||https://hdl.handle.net/10356/155221||ISSN:||1754-2189||DOI:||10.1038/s41596-020-0310-z||Rights:||© 2020 Nature Protocols. All rights reserved.||Fulltext Permission:||none||Fulltext Availability:||No Fulltext|
|Appears in Collections:||SBS Journal Articles|
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