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Department of Chemistry pres.

Pushing the boundaries of H/D exchange-mass spectrometry to analyze glycans

Elyssia Gallagher (Baylor University)

Glycans are 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 binding interactions of solvated proteins. Carbohydrates, model systems for glycans, are susceptible to HDX since they contain labile hydrogens, primarily in the form of hydroxyls, which can be labeled with deuterium (D) upon exposure to deuterated solvents. However, compared to backbone amides, the functional group detected in traditional HDX-MS experiments for proteins, the exchange rate of glycan hydroxyls is two to eight orders of magnitude faster, depending on solution pH. This rapid exchange rate makes it unfeasible to monitor HDX of carbohydrate hydroxyls using traditional, bottom-up HDX methods.
Herein, we describe our ongoing efforts to characterize solvated carbohydrates. We perform rapid HDX by introducing deuterating reagents (e.g. D2O) to carbohydrates during electrospray ionization (ESI). Our work illustrates that these rapid, in-ESI HDX methods characterize solvated carbohydrates rather than gas-phase structures. Furthermore, we have coupled our experimental work with molecular dynamics simulations to identify the mechanism of carbohydrate ionization during ESI. Experimentally, we have quantified how ESI source conditions alter the magnitude of HDX for carbohydrate model systems, developed an internal standard to control for daily humidity differences, and established methods to alter the HDX labeling time on the microsecond to millisecond timescale. We are now applying these in-ESI HDX methods to characterize carbohydrate isomers and non-covalent carbohydrate-protein interactions.



Elyssia Gallagher (Baylor University)

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