Cellular Thermal Shift Assay

Kranz and Schalk-Hihi, 2011 Kranz J.K.

Schalk-Hihi C. Protein thermal shifts to identify low molecular weight fragments. Martinez Molina et al., 2013 Martinez Molina D.

Jafari R.

Ignatushchenko M.

Seki T.

Larsson E.A.

Dan C.

Sreekumar L.

Cao Y.H.

Nordlund P. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Martinez Molina and Nordlund, 2016 Martinez Molina D.

Nordlund P. The cellular thermal shift assay: a novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies. Savitski et al. (2014) Savitski M.M.

Reinhard F.B.M.

Franken H.

Werner T.

Savitski M.F.

Eberhard D.

Molina D.M.

Jafari R.

Dovega R.B.

Klaeger S.

et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. Huber et al., 2015 Huber K.V.M.

Olek K.M.

Muller A.C.

Tan C.S.H.

Bennett K.L.

Colinge J.

Superti-Furga G. Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Wilhelm et al., 2014 Wilhelm M.

Schlegl J.

Hahne H.

Gholami A.M.

Lieberenz M.

Savitski M.M.

Ziegler E.

Butzmann L.

Gessulat S.

Marx H.

et al. Mass-spectrometry-based draft of the human proteome. Thermally induced unfolding of a purified protein can be followed by differential static light scattering or differential scanning fluorometry and often results in a sigmoidal melting curve, which enables the determination of a distinct melting temperature (Tm). Binding of a small molecule to a protein can lead to thermal stabilization or even destabilization resulting in a shift of this melting curve and consequently in a shift of Tm (ΔTm). This principle has been employed to study ligand-protein interactions and was termed thermal shift assay (TSA) (). Recently, this methodology was extended to the determination of thermal shifts in living cells using immunoblotting and was therefore called cellular thermal shift assay (CETSA). The assay employs protein aggregation upon unfolding; a behavior that is similar both for purified proteins and for proteins in living cells (). In contrast to the classical TSA, which follows the unfolding of a given protein, in a CETSA experiment, cells are heat treated followed by precipitation of the aggregated pool of the protein and quantification of the remaining soluble protein fraction by immunoblotting ( Figure 2 ). The remaining fraction corresponds to the non-denatured folded protein. Tm is then inferred by plotting the amount of soluble protein against the temperature. The most appealing advantages of this methodology lie in the employment of ligands without any chemical modifications (e.g., linker attachment), and the use of equipment that is usually available in every biochemistry lab. Several applications of this approach were recently reviewed (). However, if immunoblotting is employed for protein detection and quantification, CETSA is applicable only to ligands with known or putative targets and relies on the availability of suitable antibodies. It was realized early on that implementation of a quantitative mass spectroscopy (MS)-based setup would overcome these limitations and would allow parallel binding studies, which was first reported by Figure 2 ). Since this approach targets the whole proteome in an unbiased manner it was termed thermal proteome profiling (TPP). The parallel determination of melting curves for some 3,400 proteins, even after stringent filtering, was reported (). This is a remarkable achievement given that the human proteome comprises ca. 20,000 proteins, of which 10,000–12,000 proteins are ubiquitously expressed in all human cells ().

Figure 2 Cellular Thermal Shift Assay Show full caption (A–C) After incubation with a compound or a vehicle control, cells are aliquoted and heated to ten different temperatures. Aggregated proteins are subsequently precipitated by centrifugation. The remaining soluble protein fraction is analyzed either via mass spectrometry or immunoblotting. The mass spectrometric readout enables (A) thermal proteome profiling (TPP), the determination of (B) isothermal-dose-response relations (ITDR), or (C) time-resolved ITDR for thousands of proteins in parallel.

Franken et al., 2015 Franken H.

Mathieson T.

Childs D.

Sweetman G.M.A.

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Togel I.

Doce C.

Gade S.

Bantscheff M.

Drewes G.

et al. Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry. Reinhard et al., 2015 Reinhard F.B.M.

Eberhard D.

Werner T.

Franken H.

Childs D.

Doce C.

Savitski M.F.

Huber W.

Bantscheff M.

Savitski M.M.

et al. Thermal proteome profiling monitors ligand interactions with cellular membrane proteins. Savitski et al., 2014 Savitski M.M.

Reinhard F.B.M.

Franken H.

Werner T.

Savitski M.F.

Eberhard D.

Molina D.M.

Jafari R.

Dovega R.B.

Klaeger S.

et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. The MS-based approach uses isobaric tandem-mass-tag (TMT) labeling, which enables the simultaneous quantification of proteins under up to ten different experimental conditions. Thus, a temperature range from 37°C to about 67°C was suggested in which most proteins show sigmoidal melting curves (). This range can be covered with a 10plex TMT label with increments of about 3°C.

Savitski et al., 2014 Savitski M.M.

Reinhard F.B.M.

Franken H.

Werner T.

Savitski M.F.

Eberhard D.

Molina D.M.

Jafari R.

Dovega R.B.

Klaeger S.

et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. 50 values (negative decimal logarithm of the half-maximal inhibitory concentration) determined in kinobead assays and pEC 50 values (negative decimal logarithm of the half-maximal effective concentration) obtained with the TPP-CCR technique showed a good correlation ( Savitski et al., 2014 Savitski M.M.

Reinhard F.B.M.

Franken H.

Werner T.

Savitski M.F.

Eberhard D.

Molina D.M.

Jafari R.

Dovega R.B.

Klaeger S.

et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. TPP comes in two different flavors: (1) In the “classical” temperature range (TR) experiment ( Figure 2 A), a fixed compound concentration is used that is high enough to fully saturate the binding site of the target to achieve maximal stabilization, which results in a maximal protein Tm shift between the compound-treated and control samples. Ferrochelatase, an enzyme involved in the biosynthesis of heme, was identified as an off-target kinase inhibitor in a TPP-TR experiment, which might explain the phototoxicity of some members of this inhibitor class (). (2) An isothermal-dose response (ITDR) experiment employs a compound concentration range (CCR) at a fixed temperature ( Figure 2 B). At the respective temperature, most of the protein should be denatured and aggregated in the absence of the compound while most of the protein should still be soluble in the presence of the compound. A range of ten different concentrations can be explored due to the multiplexing properties of the TMT label. The TPP-CCR experiment allows the determination of ligand affinities in intact cells and, thus, a quantitative target engagement analysis. For the kinase inhibitor GSK3182571, a comparison between pICvalues (negative decimal logarithm of the half-maximal inhibitory concentration) determined in kinobead assays and pECvalues (negative decimal logarithm of the half-maximal effective concentration) obtained with the TPP-CCR technique showed a good correlation ().

If ITDR is performed in a time-dependent manner, even the uptake of a compound over time can be followed ( Figure 2 C). For this, TPP-CCR experiments are performed upon treatment of the cells with the ligand for different time intervals. Directly after compound administration, almost no compound is imported into the cells resulting in a low intracellular compound concentration. Consequently, target occupancy as well as protein stabilization are low. With increased incubation time, the compound concentration in the cells rises and can be measured as an increase in protein stabilization, i.e., increased ΔTm. After the maximal intracellular compound concentration is reached no further increase in ΔTm is observed.