Carbohydrates play key roles in facilitating cellular functions, yet characterizing their structures is analytically challenging due to the presence of epimers, regioisomers, and stereoisomers. In-electrospray-hydrogen/deuterium exchange-mass spectrometry (in-ESI HDX-MS) is a rapid HDX method that samples solvated carbohydrates with minimal instrument modification. When applied to proteins, HDX is often measured after multiple time points to sample the dynamics of structures. Herein, we alter the HDX reaction time by modifying the spray-solvent conductivity, which changes the initial size of ESI droplets, and thus, the droplet lifetimes. We show that this change in droplet lifetime alters the magnitude of HDX for carbohydrate-metal adducts. Furthermore, we illustrate how monitoring HDX at multiple time points enables three trisaccharide isomers (melezitose, maltotriose, and isomaltotriose) to be distinguished. This work illustrates the feasibility of this method for characterizing solvated carbohydrates, including isomeric species which differ only by linkage.

Glycans, carbohydrates, and glycoconjugates are involved in many crucial biological processes, such as disease development, immune responses, and cell–cell recognition. Glycans and carbohydrates are known for the large number of isomeric features associated with their structures, making analysis challenging compared with other biomolecules. Mass spectrometry has become the primary method of structural characterization for carbohydrates, glycans, and glycoconjugates. Metal adduction is especially important for the mass spectrometric analysis of carbohydrates and glycans. Metal‐ion adduction to carbohydrates and glycoconjugates affects ion formation and the three‐dimensional, gas‐phase structures. Herein, we discuss how metal‐ion adduction impacts ionization, ion mobility, ion activation and dissociation, and hydrogen/deuterium exchange for carbohydrates and glycoconjugates. We also compare the use of different metals for these various techniques and highlight the value in using metals as charge carriers for these analyses. Finally, we provide recommendations for selecting a metal for analysis of carbohydrate adducts and describe areas for continued research.

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This pilot study examined how exemestane (an aromatase inhibitor [AI]) affected osteoprotegerin (OPG) urine concentrations in postmenopausal women. Exemestane (25 mg, single dose) was given to 14 disease‐free women past menopause in this nonrandomized, open‐label study. Before dosing, urine specimens were gathered. Three days later, these women returned to provide urine specimens for pharmacokinetic (measurement of major parent drug and enzymatic product) and pharmacodynamic (profiling of OPG) analysis. Urine concentrations of the major parent drug (exemestane) and enzymatic product (17‐hydroexemestane) were quantified using liquid chromatography‐tandem mass spectrometry. An analyst software package was used for data processing. Following the manufacturer's guidelines, OPG urine concentrations were quantified using a human osteoprotegerin TNFRSF11b ELISA kit from Sigma‐Aldrich. A microplate reader helped to carry out OPG data analysis and processing. Our results highlight that OPG urine concentrations were decreased 3 days after drug dosage (mean predosage OPG concentration, 61.4 ± 24.1 pg/mL; vs mean postdosage OPG concentration, 45.7 ± 22.1 pg/mL; P = .02, Wilcoxon rank test). Among the 14 volunteers enrolled in the study, 4 subjects had an increase of less than 1‐fold, and the rest showed an average of a 2‐fold decrease in OPG concentration (range, 1.1‐5.4; standard deviation, 1.3) after exemestane administration. There was no association between fold decrease in OPG urine concentration and the pharmacokinetics of the major parent drug (exemestane) and its enzymatic product (17‐hydroexemestane). We concluded that one of the off‐target pharmacological effects of AIs (eg ,exemestane) may result in the reduction of osteoprotegerin.