Hydrogen-Deuterium Exchange Coupled to Top- and Middle-Down Mass Spectrometry Reveals Histone Tail Dynamics before and after Nucleosome Assembly

Until recently, a major limitation of hydrogen-deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization was limited to the length of the peptide generated during proteolysis. However, electron transfer dissociation (ETD) has been shown to preserve deuterium label in the gas phase, enabling better resolution. To date, this technology remains mostly limited to small, already well-characterized proteins. Here, we optimize, expand, and adapt HDX-MS tandem MS (MS/MS) capabilities to accommodate histone and nucleosomal complexes on top-down HDX-MS/MS and middle-down 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 analysis by traditional structural biology techniques for decades, revealing important insights into chromatin biology. 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. [Display omitted] •Middle-down and top-down hydrogen-deuterium exchange (HDX) methods were developed•HDX was applied to nucleosomal and sub-nucleosomal complexes•Histone tail dynamics were elucidated in unprecedented detail•The H4 tail is significantly protected from exchange in the nucleosome Hydrogen-deuterium exchange (HDX) is a technique to probe protein dynamics in solution. Karch et al. build upon existing HDX methodology to improve the resolution of these techniques and study histone N-terminal tail domains, which are critical for many nuclear functions including transcription.