hydrogen deuterium exchange
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2021 ◽  
Author(s):  
Neeleema Seetaloo ◽  
Monika Kish ◽  
Jonathan James Phillips

Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labelled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labelled protein (top-down), using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined - they can only be analyzed separately. Thus, we developed HDfleX - a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.


2021 ◽  
pp. 167398
Author(s):  
Kyle Trainor ◽  
Colleen M. Doyle ◽  
Avril Metcalfe-Roach ◽  
Julia Steckner ◽  
Daša Lipovšek ◽  
...  

2021 ◽  
Author(s):  
Ritu Chaturvedi ◽  
Ian Webb

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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