Fig. 2
Thermal proteome profiling (TPP) method can be performed in one of three modes: temperature range (TPP-TR); compound concentration range (TPP-CCR); or two-dimensional TPP (2D-TPP). The general procedure is composed of (1) preparation of cells for the experiment, in which either cell extracts are prepared or intact cells are cultured; (2) drug treatment with either a single compound concentration (TPP-TR) or a range of compound concentrations (TPP-CCR and 2D-TPP); (3) heating the cells to a range of temperatures (TPP-TR and 2D-TPP) or a single temperature (TPP-CCR); (4) extraction of soluble protein fraction using ultracentrifugation after cell lysis—a mild detergent can be included to solubilize membrane proteins; (5) protein digestion using a proteolytic enzyme followed by peptide labeling with neutron-encoded isobaric tags (at this step, the illustration shows an example of the procedure for a TPP-TR experiment, but an analogous labeling scheme is used for TPP-CCR or 2D-TPP—see details in the main text); (6) mass spectrometric analysis using an Orbitrap mass spectrometer to resolve the 6 mDa differences between some of the adjacent TMT reporter ions (again, at this step, the illustration shows an example of the resulting spectra of one peptide following a TPP-TR experiment); and (7) data processing to obtain plots like the ones illustrated: for TPP-TR, melting curves for each protein in the absence of presence of drug will be generated—target engagement is observed as a shift in the apparent melting temperature (Tm) of the protein; for TPP-CCR, potency curves for each protein will be obtained—from these curves it is possible to estimate the potency of the drug against each of the targets; for 2D-TPP, heat maps colored by the intensity of the abundance of soluble protein at each concentration and temperature will be generated