Thermodynamic Analysis of Protein Stability and Ligand Binding Using a Chemical Modification- and Mass Spectrometry-Based Strategy

West, Graham M.; Tang, Liangjie; Fitzgerald, Michael C.

doi:10.1021/ac702610a

Cited by 193 publications

(220 citation statements)

References 41 publications

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“…Indeed, the results in Fig. 3 are consistent with the destabilizing effects of methionine oxidation that we have previously measured in the CypA system (11). In general, SPROX data obtained on nonoxidized peptides are expected to provide the most accurate measure of a protein's wild-type stability.…”

Section: Resultssupporting

confidence: 87%

“…Whereas we have previously shown that accurate ΔΔG f values for some protein systems can be determined by SPROX, even if the protein oxidation products are destabilized (11), it is clear that not all protein-folding and ligand-binding interactions will be free from perturbation. However, as described above SPROX data obtained on nonoxidized forms of methioninecontaining peptides are expected to be useful regardless of the perturbing effects of the oxidation reaction.…”

Section: Resultsmentioning

confidence: 92%

“…1 and Fig. S1) combines a chemical modificationand mass spectrometry (MS)-based method, termed Stability of Proteins from Rates of Oxidation (SPROX) (11), with quantitative MS-based proteomics. The proteomics platform used here relied on a multidimensional protein identification technology (MudPIT) (12) and the use of isobaric mass tags (13) for peptide quantitation.…”

Section: Resultsmentioning

confidence: 99%

“…Our initial development of SPROX included a matrix-assisted laser desorption/ionization (MALDI)-and/or electrospray ionization (ESI)-based readout of the intact protein (11). The integration of SPROX into the LC-MS-based proteomics platform described here requires a peptide readout in which the disappearance of nonoxidized methionine-containing peptides and/or the appearance of oxidized methionine-containing peptides are quantified as a function of the SPROX buffer denaturant concentration.…”

Section: Resultsmentioning

confidence: 99%

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

West

Tucker

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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164

177

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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 ∼ 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.cyclosporine | liquid chromatography-mass spectrometry | ligand binding | protein folding | thermodynamics K nowledge about protein-drug interactions is critical for understanding the therapeutic effects and pleiotropic activities of drugs. Whereas genome-and proteome-based expression profiling studies of organisms and cell lines grown in the presence of drugs can provide valuable information about the functional analysis of drugs (e.g., the physiological processes and biological pathways impacted by both the on-and off-target effects of drug action), such expression profiling studies do not provide a readout of the specific protein interactions involved in a drug's mode of action. The comprehensive analysis of such drug-induced protein interactions requires the ability to assay all the proteins in a proteome for drug binding and to quantitatively assess their affinity for the drug target.Several protein ligand-binding assays, such as the yeast twohybrid assay (1, 2) and affinity chromatography techniques coupled with mass spectrometry (3-6), exist for the large-scale analysis of protein-protein interactions. However, generic protein ligand-binding assays are lacking for the detection of protein-drug interactions on the proteomic scale. Although applications of the yeast two-hybrid assay and affinity chromatography techniques to nonprotein ligands have been reported (7-9), these approaches are frequently problematic with nonprotein ligands (e.g., small drug molecules) due to the inherent difficulties associated with conjugating such ligands to the necessary bait protein in the yeast-based assay and to the necessary solid support in the affinity chromatography and mass spectrometry-based assay. Conventional protein ligand-binding assays (e.g., those based on calorimetric and spectroscopic techniques) are also not amenable to analyses on ...

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

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

West

Tucker

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

164

177

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“…[1][2][3] In these approaches, binding is detected by the ligand's ability to increase the thermodynamic stability of its protein partner. 4 By interacting preferentially with the folded conformation(s), the ligand shifts the equilibrium away from the unfolded state, which is measured in a denaturation experiment.…”

Section: Introductionmentioning

confidence: 99%

Protein–protein binding affinities by pulse proteolysis: Application to TEM‐1/BLIP protein complexes

et al. 2010

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Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the b-lactamase TEM-1 and various b-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C m , of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n 5 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and DC m was observed. From this ''calibration curve,'' accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived DC m values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies.

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Chemical Footprinting for Determining Protein Properties and Interactions

Kerfoot¹,

Gross

2011

Protein and Peptide Mass Spectrometry in Drug Discovery

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Thermodynamic Analysis of Protein Stability and Ligand Binding Using a Chemical Modification- and Mass Spectrometry-Based Strategy

Cited by 193 publications

References 41 publications

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

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

Protein–protein binding affinities by pulse proteolysis: Application to TEM‐1/BLIP protein complexes

Chemical Footprinting for Determining Protein Properties and Interactions

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