Fig. 3
From: The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin
Thermal proteome profiling (TPP) for the identification of prodigiosin targets and prodigiosin-affected proteins: GRASP55 is thermally stabilized by treatment with prodigiosin. A RTSA analysis plot of the statistical significance vs. the extent of the effect of prodigiosin on protein intensity collated over the different temperatures. Differential protein intensities may result from prodigiosin-mediated thermal protein stability alternation and/or a change in protein abundance (caused by, e.g., differential protein expression, degradation or secretion), where these two effects cannot be distinguished in the current plot. Significant proteins given by the RTSA software are colored in green or red for positive or negative RTSA distance score, respectively. B Volcano-like plot of the statistical significance vs. the extent of prodigiosin-mediated thermal protein stabilization (mean ΔTm > 0) or destabilization (mean ΔTm < 0). The color code refers to significant proteins by RTSA (see panel A). GRASP55 is the RTSA significant protein with the highest statistical significance among the stabilized proteins and, thus, a highly promising prodigiosin target protein candidate. C Volcano-like plot of the statistical significance vs. the extent of prodigiosin-mediated change in protein abundance (higher or lower for mean log2 ratio > 0 or < 0, respectively) calculated from protein intensities at 36.5 °C. The color code refers to significant proteins by RTSA (see panel A). D Melting curves (solid lines) of GRASP55 from prodigiosin or DMSO treated cells (RTSA software output of TPP-TR analysis). Datapoints and whiskers represent the arithmetic mean ± SD of three replicates. Datapoints for temperatures showing significant intensity differences are dash-boxed, next to which the collation ratio and (uncorrected) p-value are given. The thermal stabilization of GRASP55 by prodigiosin (with a mean melting point difference of 2.1 °C) is indicated by dashed lines and a measure for statistical significance is provided (-lg(p), same as y-axis of panel B). E Immunoblotting for GRASP55 protein quantification from the non-denatured protein fractions of prodigiosin or DMSO treated HeLa cells (CETSA). F Melting curves of GRASP55 from quantitative immunoblotting (CETSA, see panel E) using the same RTSA analysis and representation as for TPP-TR (see panel D). The thermal stabilization of GRASP55 by prodigiosin was confirmed