AbstractUntil recently, a major limitation of hydrogen deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization information was limited to the length of the peptide generated during proteolysis. Recently, however, it has been demonstrated that electron transfer dissociation (ETD) allows for preservation of deuterium label in the gas phase and therefore can be used to obtain more resolved information. To date, this technology has remained mostly limited to single, small, already well-characterized model proteins. Here, we optimize, expand, and adapt HDX-MS/MS capabilities to accommodate histone and nucleosomal complexes on top-down (TD) HDX-MS/MS and middle-down (MD) HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded rigorous analysis by traditional structural biology techniques for decades, revealing important novel insights into chromatin biology. This work represents the first heterogeneous protein complex and protein-DNA complex to be analyzed by TD- and MD-HDX-MS/MS, respectively. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.
Sites Involved in Intra- and Interdomain Allostery Associated with the Activation of Factor VIIa Pinpointed by Hydrogen-Deuterium Exchange and Electron Transfer Dissociation Mass Spectrometry
