Combining H–D exchange and ESI-FAIMS-MS for detecting gas-phase conformers of equine cytochrome c

2005 ◽  
Vol 83 (11) ◽  
pp. 1961-1968 ◽  
Author(s):  
Randy W Purves ◽  
Barbara Ells ◽  
David A Barnett ◽  
Roger Guevremont
Keyword(s):  
Cytochrome C ◽  
Gas Phase ◽  
Ion Mobility ◽  
Protein Conformations ◽  
Exchange Mass Spectrometry ◽  
Multiply Charged ◽  
Hydrogen Deuterium ◽  
Complementary Nature ◽  
Charged Ions ◽  
Exchange Data

Conformers of equine cytochrome c were investigated in the gas phase using a combination of high-field asymmetric waveform ion mobility spectrometry (FAIMS) and hydrogen–deuterium (H–D) exchange. Electrospray generated ions of equine cytochrome c were exposed to a low concentration of D2O vapour while being transported by a flow of nitrogen through a FAIMS device. During this transport period of about 250 ms in the FAIMS analyzer, the various conformers of multiply charged ions of cytochrome c were simultaneously undergoing H–D exchange and being separated from each other. The extent of H–D exchange was calculated from the observed m/z of conformers after exposure to D2O vapour in FAIMS. The complementary nature of these two methods resulted in observations supporting a greater number of conformers (e.g., at least 11 conformers were identified for the +16 charge state) than would be expected by analyzing the FAIMS data and the H–D exchange data independently.Key words: FAIMS, H–D exchange, mass spectrometry, protein conformations, electrospray ionization.

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.

Reactions of dimethylamine with multiply charged ions of cytochrome c

1990 ◽  
Vol 112 (14) ◽  
pp. 5668-5670 ◽  
Author(s):  
Scott A. McLuckey ◽  
Gary J. Van Berkel ◽  
Gary L. Glish
Keyword(s):  
Cytochrome C ◽  
Multiply Charged Ions ◽  
Multiply Charged ◽  
Charged Ions

Time-of-flight spectrometer for absolute measurements of multiply charged ions and ionic molecular fragments in the gas phase

2002 ◽  
Vol 73 (6) ◽  
pp. 2369-2374 ◽  
Author(s):  
A. C. F. Santos ◽  
W. S. Melo ◽  
M. M. Sant’Anna ◽  
G. M. Sigaud ◽  
E. C. Montenegro
Keyword(s):  
Gas Phase ◽  
Time Of Flight ◽  
Multiply Charged Ions ◽  
Molecular Fragments ◽  
Multiply Charged ◽  
Absolute Measurements ◽  
Charged Ions

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>

Naked Protein Conformations: Cytochrome c in the Gas Phase

1995 ◽  
Vol 117 (40) ◽  
pp. 10141-10142 ◽  
Author(s):  
David E. Clemmer ◽  
Robert R. Hudgins ◽  
Martin F. Jarrold
Keyword(s):  
Cytochrome C ◽  
Gas Phase ◽  
Protein Conformations

scholarly journals Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Structure of Herceptin®

2018 ◽  
Author(s):  
Rosie Upton ◽  
Lukasz G. Migas ◽  
Kamila J. Pacholarz ◽  
Richard G. Beniston ◽  
David Firth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Ion Mobility Mass Spectrometry ◽  
Data Sets ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Interactive Data ◽  
Hdx Ms

<p>To consider the measurable variations in biopharmaceuticals we use mass spectrometry and systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry analyses. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5 – 25 %. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data supports this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together these findings, and the supplied interactive data sets could be used to provide acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs. </p>

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