Fig. 4

GRASP55 is stabilized at low nanomolar prodigiosin concentrations. For thermal proteome profiling compound concentration range (TPP-CCR) experiments, HeLa wt cells were treated with ten different concentrations of prodigiosin for 6 h, harvested, and cell suspensions were exposed to a short (3 min) constant temperature treatment at 50 °C (or 37 °C to test for abundance effects). Cells were lysed and the non-denatured protein fraction was recovered after centrifugation followed by quantitative MS analysis as described for TPP-TR, resulting in dose response characteristics for prodigiosin-affected proteins. A Plot of the dose response curve fitting parameters pseudo-R² (proteins with a pseudo-R² value of > 0.8 were considered to have prodigiosin dose–response characteristics) vs. pEC₅₀ (the negative decadic logarithm of the half-maximal effective concentration) for proteins exhibiting increasing intensities at increasing prodigiosin concentrations (positive dose response). The data point diameter encodes the pseudo-R² (50 °C – 37 °C) difference (high for solely (de)stabilized proteins, low for solely abundance affected proteins). Proteins with pEC₅₀ > 8 (EC₅₀ < 10 nM) are labeled. GRASP55 is the protein affected (stabilized) at the lowest prodigiosin concentrations among the proteins with positive dose response. B Same representation as for panel A but for proteins with negative dose response. Lysosome associated proteins (UniProt annotated keyword KW-0458) are underlined. C Dose response characteristics and fitting results for GRASP55 at 50 °C. Data points and whiskers represent the arithmetic mean ± SD of three replicates and the fitted dose response curve is shown in red. D Same representation as in panel C but for 37 °C, with the absence of a dose–response effect (irrelevant pseudo-R2 <  < 0.8) represented by a thin dotted fit curve (in red)