Tutorial: Chemistry of Hydrogen/Deuterium Exchange Mass Spectrometry

Chemistry related to hydrogen/deuterium exchange-mass spectrometry (HDX-MS) for the analysis of proteins is described. First, the HDX rates of various functional groups in proteins are explained by reviewing the observed rates described in the literature, followed by estimating rates of all types of heteroatom hydrogens in proteins using proton transfer theory and the pK_a values. The estimated HDX rates match well with the respective observed rates for most functional groups, with the exception of indole and amide groups. The discrepancies between the observed and estimated HDX rates for these groups are explained by the reaction mechanisms. Second, the factors that affect the HDX rates of backbone amide hydrogen, including side chain, N- and C-terminals, pH, temperature, organic solvent, and isotopes, are discussed. These factors are important for the proper design of exchange reactions and downstream process as well as the analysis and interpretation of HDX-MS data.