Glycosylation is a post-translational modification that changes during cellular differentiation, immune response, and in metastatic cancerous tissue. Characterizing glycan structures is analytically challenging since glycans are heterogeneous, branched polymers with different monosaccharide isomers and linkage stereochemistries. Tandem mass spectrometry and ion mobility are powerful tools for elucidating glycan structures; however, these techniques sample glycans as gas-phase ions. Hydrogen/deuterium exchange – mass spectrometry (HDX-MS) has been used to characterize the conformations and dynamics of native, solvated biomolecules. In HDX-MS, the mass increase of an analyte is measured as labile protons are replaced with deuterium (D) upon exposure to deuterated solvents (e.g. D2O). The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. However, unlike proteins, carbohydrates and glycans contain many rapidly exchanging functional groups (e.g. hydroxyls). Here, we will compare different HDX techniques for sampling solvated carbohydrates and illustrate the use of these methods for analyses of carbohydrate structures. Following solution mixing, we observed D-uptake in carbohydrates; however, the level of D-uptake varied as a function of infusion rate into the source of the instrument. Further experiments indicated that evaporated D2O from the solvent accumulated in the source and promoted HDX. Though in-ESI HDX has been previously described for carbohydrates, our work illustrates the effect of the infusion solvent on HDX and the need to control the source environment. To utilize the source as a labeling chamber, we methodically analyzed the effects of source parameters (spray voltage, sheath and auxiliary gas flow rates) and sample conditions (solvent composition and sample concentration) on D-uptake of carbohydrates and identified a set of optimum conditions for in-ESI HDX. We developed and utilized an internal standard, maltoheptaose derivatized by reaction with Girard’s T-reagent, to increase the repeatability of HDX measurements made on multiple days. Furthermore, we measured a difference in the relative D-uptake for two trisaccharide isomers (melezitose and maltotriose), detected as sodium-adducts in the MS, indicating that this method has promise in identifying differences in carbohydrate structures.

Hydrogen/deuterium exchange – mass spectrometry of rapidly exchanging functional groups: Towards analyses of solvated glycan structures

256th National Meeting (2018)

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