Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data.

The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. Graphical Abstract ᅟ.