scholarly journals Multiplexed Conformationally-Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation

2021 ◽  
Author(s):  
Ritu Chaturvedi ◽  
Ian Webb
Keyword(s):  
Cytochrome C ◽  
Gas Phase ◽  
Charge State ◽  
Electron Capture ◽  
Electron Capture Dissociation ◽  
Ion Mobility ◽  
Transmission Mode ◽  
Hydrogen Deuterium Exchange ◽  
Protein Ions ◽  
Hydrogen Deuterium

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.

Do electrospray mass spectra reflect the ligand binding state of proteins in solution?

2005 ◽  
Vol 83 (11) ◽  
pp. 1953-1960 ◽  
Author(s):  
Belal M Hossain ◽  
Douglas A Simmons ◽  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Protein Interactions ◽  
Noncovalent Complex ◽  
Deuterium Exchange ◽  
Solution Phase ◽  
Hydrogen Deuterium Exchange ◽  
Esi Ms ◽  
Protein Ions ◽  
Hydrogen Deuterium

Electrospray ionization (ESI) mass spectrometry (MS) has become a popular tool for monitoring ligand–protein and protein–protein interactions. Due to the "gentle" nature of the ionization process, it is often possible to transfer weakly bound complexes into the gas phase, thus making them amenable to MS detection. One problem with this technique is the potential occurrence of fragmentation events during ESI. Also, some analytes tend to cluster together during ionization, thus forming nonspecific gas-phase assemblies that do not represent solution-phase complexes. In this work, we implemented a hydrogen–deuterium exchange (HDX) approach that can reveal whether or not the free and (or) bound constituents of a complex observed in ESI-MS reflect the binding situation in solution. Proteins are subjected to ESI immediately following an isotopic labeling pulse; only ligand-free and ligand-bound protein ions that were formed directly from the corresponding solution-phase species showed different HDX levels. Using myoglobin as a model system, it is demonstrated that this approach can readily distinguish scenarios where the heme–protein interactions were disrupted in solution from those where dissociation of the complex occurred in the gas phase. Experiments on cytochrome c strongly suggest that dimeric protein ions observed in ESI-MS reflect aggregates that were formed in solution.Key words: electrospray mass spectrometry, ligand–protein interaction, noncovalent complex, hydrogen–deuterium exchange, protein folding.

scholarly journals Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Travelling Wave Device

2018 ◽  
Author(s):  
Charles Eldrid ◽  
Jakub Ujma ◽  
Symeon Kalfas ◽  
nick tomczyk ◽  
Kevin Giles ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Gas Phase ◽  
Ion Mobility ◽  
Structural Changes ◽  
Structural Information ◽  
Travelling Wave ◽  
Resolving Power ◽  
Small Proteins ◽  
Multimeric Complex ◽  
Protein Ions

<div>Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into “conformational families”. Increasing the IM resolving power should allow finer structural information to be obtained, and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds. Here, we report on investigations of protein ion stability using a multi-pass travelling wave (TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A). The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed which were used to obtain more detailed collision induced unfolding pathways for Cytochrome C. The novel instrument geometry provide unique capabilities with the potential to expand the field of protein analysis via IM-MS.</div>

scholarly journals Comparison of Peptide Ion Conformers Arising from Non-Helical and Helical Peptides Using Ion Mobility Spectrometry and Gas-Phase Hydrogen/Deuterium Exchange

2018 ◽  
Vol 29 (12) ◽  
pp. 2402-2412 ◽  
Author(s):  
Ahmad Kiani Karanji ◽  
Mahdiar Khakinejad ◽  
Samaneh Ghassabi Kondalaji ◽  
Sandra N. Majuta ◽  
Kushani Attanayake ◽  
...  
Keyword(s):  
Gas Phase ◽  
Ion Mobility Spectrometry ◽  
Ion Mobility ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Helical Peptides ◽  
Hydrogen Deuterium
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