Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Karch KR

PROVIDER: S-EPMC6281757 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Karch Kelly R KR Coradin Mariel M Zandarashvili Levani L Kan Zhong-Yuan ZY Gerace Morgan M Englander S Walter SW Black Ben E BE Garcia Benjamin A BA

Structure (London, England : 1993) 20181004 12

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 ...[more]

PMID: 30293810

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Project description:Hydrogen exchange in solution combined with ion fragmentation in the gas phase followed by MS detection emerged in recent years as a powerful tool to study higher order protein structure and dynamics. However, a certain type of ion chemistry in the gas phase, namely, internal rearrangement of labile hydrogen atoms (the so-called hydrogen scrambling), is often cited as a factor limiting the utility of this experimental technique. Although several studies have been carried out to elucidate the roles played by various factors in the occurrence and the extent of hydrogen scrambling, there is still no consensus as to what experimental protocol should be followed to avoid or minimize it. In this study we employ fragmentation of mass-selected subpopulations of protein ions to assess the extent of internal proton mobility prior to dissociation. A unique advantage of tandem MS is that it not only provides a means to map the deuterium content of protein ions whose overall levels of isotope incorporation can be precisely defined by controlling the mass selection window, but also correlates this spatial isotope distribution with such global characteristic as the protein ion charge state. Hydrogen scrambling does not occur when the charge state of the precursor protein ions selected for fragmentation is high. Fragment ions derived from both N- and C-terminal parts of the protein are equally unaffected by scrambling. However, spatial distribution of deuterium atoms obtained by fragmenting low-charge-density protein ions is consistent with a very high degree of scrambling prior to the dissociation events. The extent of hydrogen scrambling is also high when multistage fragmentation is used to probe deuterium incorporation locally. Taken together, the experimental results provide a coherent picture of intramolecular processes occurring prior to the dissociation event and provide guidance for the design of experiments whose outcome is unaffected by hydrogen scrambling.

Dataset Information

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

Publications

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

Similar Datasets

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