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ABSTRACT: The detection and quantification of protein-ligand binding interactions is crucial in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g., drugs such as tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (isobaric mass tags for relative and absolute quantification, or iTRAQ) with a covalent labeling technique, termed stability of proteins from rates of oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g., guanidine hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand-binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to the detection of both the on- and off-target effects of ligand binding. The protocol can be completed in 5 d.
Nature Protocol 12/2012; 8(1):148-61. · 8.36 Impact Factor
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ABSTRACT: Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.
Journal of Proteome Research 09/2011; 10(11):4948-58. · 5.11 Impact Factor
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ABSTRACT: Described here is a mass spectrometry-based covalent labeling protocol that utilizes the amine reactive reagent, s-methyl thioacetimidate (SMTA), to study the chemical denaturant-induced equilibrium unfolding/refolding properties of proteins and protein-ligand complexes in solution. The protocol, which involves evaluating the rate at which globally protected amine groups in a protein are modified with SMTA as a function of chemical denaturant concentration, is developed and applied to the analysis of eight protein samples including six purified protein samples (ubiquitin, BCAII, RNaseA, 4OT, and lysozyme with, and without GlcNAc), a five-protein mixture comprised of ubiquitin, BCAII, RNaseA, Cytochrome C, and lysozyme, and a yeast cell lysate. In ideal cases the folding free energies of proteins and the dissociation constants of protein-ligand complexes can be accurately evaluated using the protocol. A direct MALDI-TOF readout is demonstrated for analysis of purified protein samples. Bottom-up proteomic strategies involving gel-based and/or LC-MS-based shotgun proteomic platforms are also demonstrated for the analyses of complex protein samples. Analysis of proteins in a yeast cell lysate suggests the SMTA-labeling protocol expands the peptide and protein coverage in chemical modification- and shotgun proteomics-based strategies for making thermodynamic measurements of protein folding and stability on the proteomic scale.
Analytical Chemistry 04/2011; 83(9):3555-62. · 5.86 Impact Factor
