Characterizing carbohydrate conformations: An application of H/D exchange – mass spectrometry with molecular dynamics simulations

Friday, March 29, 2019

Prof. Elyssia S. Gallagher

Department of Chemistry & Biochemistry

Baylor University

Glycans are structurally complex molecules with different carbohydrate subunits, linkage stereochemistries, and branching patterns; all of which play a role in their biological functions. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has become a standard method for analyzing conformations and solution dynamics of biological molecules. Carbohydrates are susceptible to HDX since they contain labile hydrogens, e.g. hydroxyls, which can be labeled with deuterium (D) upon exposure to deuterated solvents. However, the exchange rate of hydroxyls is two to eight orders of magnitude faster than protein backbone amides, depending on solution pH. This rapid exchange rate makes it unfeasible to monitor HDX of carbohydrates using traditional, bottom-up HDX methods.
Here, we will discuss our approach for sampling carbohydrate structures using rapid HDX. We introduce deuterating reagents to carbohydrates during electrospray ionization (ESI) and/or ESI droplet evaporation. We have characterized how source parameters and samples conditions affect the extent of deuterium exchange. We have also developed an internal standard to control for daily humidity differences that alter back exchange, improving measurement repeatability on different days. In the presence of this internal standard, we can distinguish differences in the extent of deuterium exchange for a pair of trisaccharide isomers. Finally, we are coupling our experimental work with molecular dynamics simulations to identify the mechanism of carbohydrate ionization and to illustrate if, and to what extent, ESI alters carbohydrate conformations compared to native, biological structures. This work will establish a foundation for applying these methods to biological glycans.

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