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

Abstract

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 DO, 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. Graphical Abstract ᅟ.

Interlaboratory Comparison of Hydrogen–Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb

“…Knowledge the overall effect of experimental factors will guide evaluation of the measurement uncertainty. One possible error could arise if the deuterium content of peptides varies as a function of charge. , Thus, deuterium content of centroids versus peptide charge was examined for 513 peptides ( z = +1 to +7) of 106 sequences reported by 13 laboratories. No evidence of systemic, in-ESI source, intermolecular H/D exchange was observed (Table S6).…”

Distinguishing Carbohydrate Isomers with Rapid Hydrogen/Deuterium Exchange-Mass Spectrometry

“…ESI parameters were spray voltage: 3.5 kV, sheath gas: 12 arb units, auxiliary gas: 0 arb units, and capillary temperature 300 °C. Between runs, the source was kept open for 2 min to prevent solvent vapor build-up . Undeuterated spectra were collected with neither the metal plate nor D 2 O droplet inside the ESI source.…”

Analysis of 16O/18O and H/D Exchange Reactions between Carbohydrates and Heavy Water Using High-Resolution Mass Spectrometry

“…Application of the hydrogen/deuterium exchange for investigating carbohydrates was previously reported [ 17 , 18 , 19 , 20 ]. E. Gallagher showed that it was possible to distinguish three different carbohydrate isomers using the H/D exchange reaction [ 21 ].…”