Quantitative proteomics approach for identifying protein-drug interactions in complex mixtures using protein stability measurements

Abstract

Knowledge about the protein targets of therapeutic agents is critical for understanding drug mode of action. Described here is a mass spectrometry-based proteomics method for identifying the protein target(s) of drug molecules that is potentially applicable to any drug compound. The method, which involves making thermodynamic measurements of protein-folding reactions in complex biological mixtures to detect protein-drug interactions, is demonstrated in an experiment to identify yeast protein targets of the immunosuppressive drug, cyclosporin A (CsA). Two of the ten protein targets identified in this proof of principle work were cyclophilin A and UDP-glucose-4-epimerase, both of which are known to interact with CsA, the former through a direct binding event (K(d) approximately 70 nM) and the latter through an indirect binding event. These two previously known protein targets validate the methodology and its ability to detect both the on- and off-target effects of protein-drug interactions. The other eight protein targets discovered here, which include several proteins involved in glucose metabolism, create a new framework in which to investigate the molecular basis of CsA side effects in humans.

Fig. 1.

Schematic representation of the experimental strategy developed in this work. A complex biological mixture of proteins (e.g., a cell lysate) is subjected to two analyses, one in the absence of drug and one in the presence of drug. In each analysis, aliquots of the protein mixture (or the protein mixture combined with drug) are diluted into a series of buffers containing increasing concentrations of a chemical denaturant [e.g., guanidinium hydrochloride (GdmCl)]. The protein samples in each GdmCl-containing buffer are reacted with the same amount of hydrogen peroxide for the same amount of time. The reaction time and concentration of hydrogen peroxide are tuned such that the thioether groups in the side chain of methionine residues are selectively oxidized. The protein oxidation reaction is quenched and the oxidized protein samples are submitted to a quantitative proteomic analysis and the nonoxidized and oxidized methionine-containing peptides are quantified as a function of the SPROX buffer denaturant concentration.

Fig. 3.

SPROX data obtained on a selected methionine-containing peptide hit from CypA, which was identified as CsA target. (A) and (B) Data obtained for the nonoxidized and oxidized (respectively) forms of the peptide. Data obtained from analyses conducted in the absence and in the presence of CsA are shown in blue and red bars, respectively. The hashed bars represent points that were missing from the data and assigned a value that was the average of the experimentally determined normalized reporter ion intensity on either side of the missing denaturant concentration. In this case the missing data was a result of poor reporter ion intensities in one of the MudPIT runs. The arrows indicate cases where there was a significant difference (> 0.6) between the normalized TMT reporter ion ratios obtained with and without CsA. The dotted lines mark the SPROX transition midpoints, which were significantly shifted (> 0.5 M) to higher denaturant concentrations in the presence of ligand.

Fig. 2.

SPROX data obtained on a selected methionine-containing peptide “nonhit” from glycerol dehydrogenase, which was not identified as a CsA target. (A) and (B) Data obtained on the nonoxidized and oxidized (respectively) forms of the peptide. Data obtained from analyses conducted in the absence and in the presence of CsA are shown in blue and red bars, respectively. The dotted lines mark the SPROX transition midpoints, which were all within a small range, < 0.5 M, and within experimental error.

Fig. 4.

Distribution of the normalized reporter ion intensity differences obtained by subtracting the normalized reporter ion intensities of the 886 methionine-containing peptides (nonoxidized and oxidized) in the absence of ligand from those intensities obtained in the presence of ligand. Approximately 85% of the observed differences were > -0.6 and < 0.6. The differences outside this range were deemed significant.