The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine. We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity.

Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Although such analyses typically assume that a protein's peptide fragments are observed with equal likelihood, only a few so-called 'proteotypic' peptides are repeatedly and consistently identified for any given protein present in a mixture. Using >600,000 peptide identifications generated by four proteomic platforms, we empirically identified >16,000 proteotypic peptides for 4,030 distinct yeast proteins. Characteristic physicochemical properties of these peptides were used to develop a computational tool that can predict proteotypic peptides for any protein from any organism, for a given platform, with >85% cumulative accuracy. Possible applications of proteotypic peptides include validation of protein identifications, absolute quantification of proteins, annotation of coding sequences in genomes, and characterization of the physical principles governing key elements of mass spectrometric workflows (e.g., digestion, chromatography, ionization and fragmentation).

The direct detection of drug-protein interactions in living cells is a major challenge in drug discovery research. Recently, we introduced an approach termed thermal proteome profiling (TPP), which enables the monitoring of changes in protein thermal stability across the proteome using quantitative mass spectrometry. We determined the intracellular thermal profiles for up to 7,000 proteins, and by comparing profiles derived from cultured mammalian cells in the presence or absence of a drug we showed that it was possible to identify direct and indirect targets of drugs in living cells in an unbiased manner. Here we demonstrate the complete workflow using the histone deacetylase inhibitor panobinostat. The key to this approach is the use of isobaric tandem mass tag 10-plex (TMT10) reagents to label digested protein samples corresponding to each temperature point in the melting curve so that the samples can be analyzed by multiplexed quantitative mass spectrometry. Important steps in the bioinformatic analysis include data normalization, melting curve fitting and statistical significance determination of compound concentration-dependent changes in protein stability. All analysis tools are made freely available as R and Python packages. The workflow can be completed in 2 weeks.

The two tandem bromodomains of the BET proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1 whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET targeted therapies.

Adenosine triphosphate (ATP) plays fundamental roles in cellular biochemistry and was recently discovered to function as a biological hydrotrope. Here, we use mass spectrometry to interrogate ATP-mediated regulation of protein thermal stability and protein solubility on a proteome-wide scale. Thermal proteome profiling reveals high affinity interactions of ATP as a substrate and as an allosteric modulator that has widespread influence on protein complexes and their stability. Further, we develop a strategy for proteome-wide solubility profiling, and discover ATP-dependent solubilization of at least 25% of the insoluble proteome. ATP increases the solubility of positively charged, intrinsically disordered proteins, and their susceptibility for solubilization varies depending on their localization to different membrane-less organelles. Moreover, a few proteins, exhibit an ATP-dependent decrease in solubility, likely reflecting polymer formation. Our data provides a proteome-wide, quantitative insight into how ATP influences protein structure and solubility across the spectrum of physiologically relevant concentrations.