In this article, we present an approach
for conformationally multiplexed,
localized hydrogen deuterium exchange (HDX) of gas-phase protein ions
facilitated by ion mobility (IM) followed by electron capture dissociation
(ECD). A quadrupole-IM-time of flight instrument previously modified
to enable ECD in transmission mode (without ion trapping) immediately
following a mobility separation was further modified to allow for
deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was
minimized to prevent deuterium scrambling from giving structurally
irrelevant results. Gas-phase HDX with ECD fragmentation for exchange
site localization was demonstrated with the extensively studied protein
folding models ubiquitin and cytochrome c. Ubiquitin
was ionized from conditions that stabilize the native state and conditions
that stabilize the partially folded A-state. IM of deuterated ubiquitin
6+ ions allowed the separation of more compact conformers
from more extended conformers. ECD of the separated subpopulations
revealed that the more extended (later arriving) conformers had significant,
localized differences in the amount of HDX observed. The 5+ charge state showed many regions with protection from HDX, and the
11+ charge state, ionized from conditions that stabilize
the A-state, showed high levels of deuterium incorporation throughout
most of the protein sequence. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with
unstructured regions of the protein relative to interior, structured
regions, especially those involved in heme binding. With careful tuning
and attention to deuterium scrambling, our approach holds promise
for determining region-specific information on a conformer-selected
basis for gas-phase protein structures, including localized characterizations
of ligand, epitope, and protein–protein binding.
Absolute K-shell ionization cross sections were measured for Ti, Co, Ge, Rb, and Sn for incident oxygen ions from 16-44 MeV. The x-rays were measured with a high resolution Si(Li) detector (166 eV at 5.9 keV). All of the data represents cross section measurements for thin targets. The measured cross sections for these elements are compared to the theoretical predictions of the Binary Encounter Approximation (BEA). Kα/Kβ ratios and energy shifts were also extracted from the data. The experimental data are compared to measured cross sections for other elements to give an overview of the systematics for oxygen ion induced x-ray production cross sections in this energy range. Some comment will also be given in regard to the use of oxygen ions to measure the parameters associated with ion implanted semiconductors.
In this article, we present an approach for conformationally multiplexed localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-ion mobility-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. This arrangement was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially-folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed greater protection against HDX than the compact 6+ conformer, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed much greater deuterium incorporation. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with exterior and more unstructured regions of the protein, while interior, structured regions, especially those involved in heme binding, were more protected against exchange. These results, as well as potential future methods and experiments, are discussed herein.
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