Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii
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Herneisen_BioProtocols_Dspace.pdf
Description
Accepted version
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873.63 KB
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Checksum (MD5)
25e9916425b57bd121a90962e3238071
Author(s)
Herneisen, Alice
Lourido, Sebastian
Date Issued
2021
Journal
BIO-PROTOCOL
Publisher
Bio-Protocol, LLC
Citation
Herneisen, Alice and Lourido, Sebastian. 2021. "Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii." BIO-PROTOCOL, 11 (21).
Version
Author's final manuscript
Abstract
Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the pathways and molecular mechanisms perturbed by such compounds requires target deconvolution. In parasites, such deconvolution has been achieved via chemogenomic approaches-for example, directed evolution followed by whole-genome sequencing or genome-wide knockout screens. As a proteomic alternative that directly probes the physical interaction between compound and protein, thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), recently emerged as a method to identify small molecule-target interactions in living cells and cell extracts in a variety of organisms, including unicellular eukaryotic pathogens. Ligand binding induces a thermal stability shift-stabilizing or destabilizing proteins that change conformationally in response to the ligand-that can be measured by mass spectrometry (MS). Cells are incubated with different concentrations of ligand and heated, causing thermal denaturation of proteins. The soluble protein is extracted and quantified with multiplexed, quantitative MS, resulting in thousands of thermal denaturation profiles. Proteins engaging the ligand can be identified by their compound-dependent thermal shift. The protocol provided here can be used to identify ligand-target interactions and assess the impact of environmental or genetic perturbations on the thermal stability of the proteome in T. gondii and other eukaryotic pathogens. Graphic abstract: Thermal proteome profiling for target identification in the apicomplexan parasite T. gondii.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.21769/BIOPROTOC.4207
