Background

Target deconvolution is a major challenge for the wealth of compounds identified through phenotypic screening. Chemogenomic approaches, such as directed evolution or drug screens, have been the favored tools for target identification in eukaryotic parasites (Paquet et al., 2017; Cowell et al., 2018; Luth et al., 2018; Rosenberg et al., 2019; Harding et al., 2020). Such approaches require culturing parasites and host cells under compound treatment for extended periods and often identify pathways indirectly affected by a small molecule rather than the target itself. In contrast, several proteomic methods developed in the past decade directly identify interactions between compounds and protein targets (McClure and Williams, 2018; Conway et al., 2021). For example, enrichment of interacting proteins can be performed with derivatized compounds for affinity-purification followed by mass spectrometry (MS). However, these approaches require specialized chemistry and introduce a linker and other chemical groups to the compound of interest, which may affect its behavior.

Thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), offers a label-free approach that can be performed in a variety of formats that preserve cellular physiology, including in situ (Dai et al., 2019; Mateus et al., 2020b). Interactions with a target are identified by a compound-dependent shift in the protein’s thermal profile. Cells or cell extracts are treated with the compound and heated to induce thermal denaturation. Aggregated proteins are removed, and soluble proteins are quantified by MS to generate melting curves for each protein. TPP has recently identified the targets of antiparasitic compounds in the apicomplexan parasites Plasmodium falciparum (Dziekan et al., 2019; Lu et al., 2020) and Toxoplasma gondii (Herneisen et al., 2020), as well as in the trypanosome Leishmania donovani (Corpas-Lopez et al., 2019).

The application of TPP extends beyond target deconvolution (Becher et al., 2018; Dai et al., 2018 and 2019; Sridharan et al., 2019; Mateus et al., 2020b). Alterations to protein state and stability may arise from conformational changes, post-translational modifications, altered localization, and interactions with other proteins and biomolecules such as metabolites and nucleic acids. For example, we performed TPP on parasites lacking mitochondrial DegP2 to identify proteins with altered stability based on the loss of this protease (Harding et al., 2020). Genetic perturbations in conjunction with functional proteome profiling are in the early stages (Mateus et al., 2020a) and may be especially well-suited to map the unannotated parts of parasite proteomes.

While TPP has been performed predominantly in mammalian systems, it is expanding to other organisms (Corpas-Lopez et al., 2019; Dziekan et al., 2019; Volkening et al., 2019; Lu et al., 2020; Herneisen et al., 2020; Harding et al., 2020; Jarzab et al., 2020). We believe this approach pairs particularly well with the study of eukaryotic parasites, whose evolutionary divergence complicates identifying molecular pathways by genomic annotation or bioinformatic analysis. For that reason, we provide a detailed protocol describing our thermal profiling pipeline developed for the organism T. gondii. Below, we identify key considerations for selecting a TPP workflow appropriate for the researcher’s biological question. Step-by-step guidelines follow.

Types of experiment

In this protocol, we stratify steps by choice of material and treatment. TPP can be performed on live parasites or parasite lysates and by melting samples over a range of 10 temperatures (“temperature range”) or over a range of 10 compound concentrations melted at a single temperature (“concentration range”). These variations give rise to four permutations described in the Procedure as (B) Lysate Temperature Range Experiment, (C) Parasite Temperature Range Experiment, (D) Lysate Concentration Range Experiment, and (E) Parasite Concentration Range Experiment. Each experiment has advantages and disadvantages (Franken et al., 2015; Dai et al., 2019; Mateus et al., 2020b). For example, experiments using live cells are more physiological but combine direct and indirect effects. Lysate experiments may more directly identify ligand-protein interactions, but loss of cellular compartmentalization can also lead to non-physiological interactions. Concentration range experiments yield more information-rich thermal profiles, but real interactions may be missed if the thermal challenge temperatures are suboptimal (too low or too high) and the overall coverage of the proteome is reduced due to global denaturation.

Treatment conditions

We have performed thermal profiling experiments on extracellular parasites to avoid the added complexity of the host proteome and confounding effects from compound permeability and host metabolism. Compound treatments are often performed on intracellular parasites; however, the appropriate concentration of compound for the thermal profiling experiment should be determined by assays using extracellular parasites. The thermal profiling experiment should mimic assay conditions as closely as possible. Considerations include the amount of time needed for the compound to arrive at diffusion and binding equilibria, the buffer in which the equilibration takes place, and equilibration temperature (e.g., room temperature vs. 37°C). Mammalian studies have often performed the incubation in PBS (Reinhard et al., 2015; Savitski et al., 2014; Franken et al., 2015). We have used a buffer composition resembling the ionic makeup of the host cytosol (Herneisen et al., 2020; Harding et al., 2020). Buffers should lack serum, which would overwhelm parasite signals that can be quantified by MS.

We aim to process 25 µg of protein per reference sample, which in our experience corresponds to the material from 1 × 10⁷ extracellular parasites of the type I RH strain. We subject concentration-range samples to at least two different thermal challenge temperatures; we have found 54°C and 58°C to work well while still providing sufficient coverage of the proteome. The thermal challenge temperatures may need to be optimized for each experiment; further commentary is provided in the Data analysis section.

Lysis conditions

The final lysis buffer composition should contain 0.5-1% IGEPAL CA-630 (also known as NP-40), which provides a balance between solubilizing membrane proteins without re-solubilizing aggregated proteins (Reinhard et al., 2015). For most experiments, the lysis buffer should contain protease inhibitors (and optionally phosphatase inhibitors, depending on the focus of the experiment) and benzonase for digestion of nucleic acids prior to the SP3 cleanup. If the compound of interest is thought to affect proteases, phosphatases, or nucleic acid binding activity, then these supplements should be omitted until after the Separation of Soluble and Aggregated Protein. Our lysis buffers have had an ionic composition similar to PBS (Herneisen et al., 2020) and an intracellular-like buffer (Harding et al., 2020), depending on the application. The ionic composition of the buffer (e.g., presence of ATP and metabolites) can substantially influence the melting behavior of proteins (Lim et al., 2018; Sridharan et al., 2019). The concentration of parasite lysate also influences melting behavior; therefore, it is crucial to count the number of parasites prior to lysis and use a consistent lysis buffer volume for the number of parasites. Following harvest, parasites should be resuspended at least once in a wash buffer that is similar in composition to the lysis buffer (but lacking detergents) to dilute cell culture contaminants, such as serum proteins.