Xu Y, et al. (2011) Mass spectrometry- and lysine amidination-based protocol for thermodynamic analysis of protein folding and ligand binding interactions. Anal Chem 83(9):3555-62
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 suggest 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.
|Status: Published||Type: Journal Article||PubMed ID: 21456597|
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