Hydrogen-Deuterium Exchange Mass Spectrometry
Mass spectrometry combined with hydrogen/deuterium (H/D) exchange is used to study the conformation and dynamics of proteins. The process is depicted schematically in the figure. When a protein is diluted into a solution of D2O, labile hydrogen atoms will exchange with deuterium. Labile protons, such as those on primary amines, exchange nearly instantaneously. Amide hydrogens exchange at rates that are characteristic of local backbone conformation and dynamics. In highly dynamic unstructured regions, the exchange reaction proceeds on the msec-sec timescale while amides that are hydrogen bonded will exchange more slowly (minutes to days). The degree of protection is indicative of local structure and dynamics.
In the MS-based approach, the exchange reaction is quenched by acidification to pH ~2.5. Quenching is followed by rapid proteolysis (usually with pepsin), to produce short peptide segments; chromatographic separation; and mass analysis. The quenching step serves to “freeze in” the pattern of deuteration imprinted on amide backbone under the deuterium labeling conditions allowing read-out of deuterium uptake to proceed under non-physiologic conditions (HPLC and MS). By monitoring the kinetics of the exchange process with MS, details about the localized backbone structure and dynamics can be determined.
The technique can be used in a comparative manner to identify changes in conformation and dynamics induced by, for example, ligand binding, changes in formulation, or point mutations.
- large proteins (> 100 kDa) and protein complexes
- complex buffer systems
- proteins that cannot be crystalized for structural studies
- proteins that have solubility problems in NMR studies
- minimal sample consumption (< 50 μg for a 50 kDa protein)
- fully automated H/D-MS system with robotic sample preparation
- state-of-the art software for data analysis
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Link to Weis Department Web page
Dr. David Weis
Department of Chemistry
1251 Wescoe Hall Drive
University of Kansas
Lawrence, KS 66045