title: Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry.
abstract: Top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection has recently matured to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. However, the scope of the proteins amenable to the analysis by top-down HDX MS still remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. While the limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace, the presence of the disulfide linkages remains a much less forgiving limitation even for the proteins of relatively modest size. To circumvent this problem, we introduce an online chemical reduction step following completion and quenching of the HDX reactions and prior to the top-down MS measurements of deuterium occupancy of individual backbone amides. Application of the new methodology to the top-down HDX MS characterization of a small (99 residue long) disulfide-containing protein β2-microglobulin allowed the backbone amide protection to be probed with nearly a single-residue resolution across the entire sequence. The high-resolution backbone protection pattern deduced from the top-down HDX MS measurements carried out under native conditions is in excellent agreement with the crystal structure of the protein and high-resolution NMR data, suggesting that introduction of the chemical reduction step to the top-down routine does not trigger hydrogen scrambling either during the electrospray ionization process or in the gas phase prior to the protein ion dissociation.
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , 710 North Pleasant Street, LGRT 104, Amherst, Massachusetts 01003 United States.
authors.forename: Guanbo
authors.initials: G
authors.lastname: Wang
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D000595:Amino Acid Sequence; D003903:Deuterium / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004220:Disulfides; D011506:Proteins; D006859:Hydrogen; D003903:Deuterium