Discussion

Proteins complexed to ligands tend to become more resistant against heat-induced unfolding. The recently developed cellular thermal shift assay (CETSA) and isothermal dose response (ITDR) approaches utilize this mechanism to assess target engagement in living cells [1]. Combining CETSA and ITDR with multiplexed quantitative mass spectrometry we generalized this concept to a proteomics scale and established the proteome-wide determination of protein thermal stability in intact cells.

Comparing the thermal profiles of up to 7,000 proteins derived from cells cultured in the presence or absence of a drug, we identified direct and indirect drug targets in living cells in an unbiased manner by large scale thermal proteome profiling (TPP) [2].
To perform bioinformatic analysis of quantified proteins from TPP experiments and identify proteins that show a significant change in their thermal stability we developed the freely available Bioconductor package “TPP”.

Applying this tool we monitored the target engagement of a large cohort of known targets of the pan-kinase inhibitor staurosporine in human K562 cells and additionally detected several hitherto unknown non-kinase targets, such as Ferrochelatase.
TPP experiments on K562 cells treated with the histone deacetylase (HDAC) inhibitor panobinostat demonstrate its effect on several known HDAC targets and also show a hitherto unknown effect on the tetratricopeptide repeat protein 38 (TTC38) [3].
In a very recent study we expanded the scope of TPP to membrane proteins by performing mild detergent extraction on Jurkat cells.

[1] Molina et al Science 2013
[2] Savitski et al Science 2014
[3] Franken et al Nature Protocols 2015 (accepted, in production)