Hydrogen/deuterium exchange mass spectrometry with high spatial resolution using gas-phase fragmentation

Daniel T. Weltz Wollenberg

Research output: ThesisPh.D. thesis

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The function of a protein is intimately linked to the intrinsic dynamics of its structure. Therefore, to fully understand the ubiquitous biological machinery that is proteins, we must probe these elusive motions. Hydrogen/deuterium exchange (HDX) of protein backbone amides provide a sensitive probe for these
structural dynamics. As each exchange of one isotope for the other incurs a change in the mass of the protein, mass spectrometry (MS) can be used to monitor this exchange in a methodology termed HDXMS. Thus, HDX-MS is a powerful tool for the study of protein structural dynamics.

While each amino acid position along the protein backbone (except for proline) provides a probe for hydrogen/deuterium exchange, the resolution offered by HDX-MS is limited to the ability to generate unique backbone fragments which can be used to infer the isotopic occupancy (1H i.e. hydrogen or 2H i.e. deuterium) for a single amide or a range of amides. For this reason, gas-phase fragmentation has been investigated as a means of achieving high resolution amide exchange data for the past two decades. However, a prerequisite for the use of gas-phase fragmentation in HDX-MS is the conservation of the solution phase isotopic labeling into the gas-phase, during gas-phase transport, and during the fragmentation process itself.
This is non-trivial due to the risk of internal energy buildup during these processes which can cause intermolecular proton migration, thus essentially randomizing the isotopic labeling of the analyte ion. This process is called hydrogen scrambling. To reduce hydrogen scrambling prior to fragmentation the energy
imparted during gas-phase ion transport must be reduced. However, this optimization generally occurs at a cost to ion transmission.

In Manuscript I enclosed in this thesis we investigate how an optimum between low hydrogen scrambling and transmission efficiency can be found at minimal cost to transmission efficiency. We also demonstrate the utility of this approach by performing bottom-up HDX-MS/MS using electron transfer dissociation
(ETD) on a well-characterized protein.

In addition to ion transport, hydrogen scrambling can be induced during the fragmentation process itself. Currently, ETD and electron capture dissociation (ECD) are well established as techniques which do not cause hydrogen scrambling in protonated peptides. More recently, this feature was also demonstrated for 213 nm ultraviolet photodissociation (UVPD) on a unique custom-build instrumental setup. Therefore, in Manuscript II enclosed in the thesis, we set out to replicate these results on the first commercial implementation of UVPD. However, to our surprise, all UVPD generated fragment ions in our commercial setup showed extensive hydrogen scrambling.
Translated title of the contributionHydrogen/deuterium udveksling målt med massespektrometri med høj rumlig opløsning ved brug af gas-fase fragmentering
Original languageEnglish
Awarding Institution
  • University of Southern Denmark
  • Jørgensen, Thomas J.D., Principal supervisor
  • Jørgensen, Christian Isak, Co-supervisor, External person
  • Mouritsen, Jeppe Christian, Co-supervisor, External person
  • Pengelley, Stuart, Co-supervisor, External person
External participants
Date of defence26. Aug 2022
Publication statusPublished - 13. Sept 2022


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