Mass Spectral Detection of Forward- and Reverse-Hydrogen/Deuterium Exchange Resulting from Residual Solvent Vapors in Electrospray Sources

Characterizing glycans is analytically challenging since glycans are heterogeneous, branched polymers with different three-dimensional conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to analyze native conformations and dynamics of biomolecules by measuring the mass increase of analytes as labile protons are replaced with deuterium following exposure to deuterated solvents. The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. HDX-MS of carbohydrates is challenging due to the rapid exchange rate of hydroxyls. Here, we describe an observed HDX reaction associated with residual solvent vapors saturating electrospray sources. When undeuterated melezitose was infused after infusing D₂O, samples with up to 73% deuterium exchange were detected. This residual solvent HDX was observed for both carbohydrates and peptides in multiple instruments and dependent on sample infusion rate, infusion time, and deuterium content of the solvent. This residual solvent HDX was observed over several minutes of sample analysis and persisted long enough to alter the measured deuterium labeling and possibly change the interpretation of the results. This work illustrates that residual solvent HDX competes with in-solution HDX for rapidly exchanging functional groups. Thus, we propose conditions to minimize this effect, specifically for top-down, in-electrospray ionization, and quench-flow HDX experiments. .