title: Mass spectrometry- and lysine amidination-based protocol for thermodynamic analysis of protein folding and ligand binding interactions.
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.
authors: Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.
authors: Ying
authors: Y
authors: Xu
authors: Irene N
authors: IN
authors: Falk
authors: Mark A
authors: MA
authors: Hallen
authors: Michael C
authors: MC
authors: Fitzgerald
mesh_terms: D000578:Amidines / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D008024:Ligands; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics; D013816:Thermodynamics
chemical_list: D000578:Amidines; D008024:Ligands; D011506:Proteins; D008239:Lysine