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Carbohydrates are critical for cellular functions as well as an important class of metabolites. Characterizing carbohydrate structures is a difficult analytical challenge due to the presence of isomers. In-electrospray – hydrogen/deuterium exchange – mass spectrometry (in-ESI HDX-MS) is a method of HDX that samples the solvated structure of carbohydrates during the ESI process and requires little to no instrument modification. Traditionally, solution-phase HDX is utilized with proteins to sample conformational differences, and pH is a critical parameter to monitor and control due to the presence of both acid- and base-catalyzed mechanisms of exchange. For In-ESI HDX, the pH surrounding the analyte changes before and during labeling, which has the potential to affect the rate of labeling for analytes. Herein, we alter the pH of spray solutions containing model carbohydrates and peptides, perform in-ESI HDX-MS, and characterize the deuterium-uptake trends. Varying pH results in altered D uptake, though the overall trends differ from the expected bulk-solution trends due to the electrospray process. These findings show the utility of varying pH prior to in-ESI HDX-MS for establishing different extents of HDX as well as distinguishing labile functional groups that are present in different analytes. 

Tandem mass spectrometry (MS/MS) using fragmentation has become one of the most effective methods for gaining sequence and structural information of biomolecules. Ion/ion reactions are competitive reactions where either proton transfer (PT) or electron transfer (ET) can occur from interactions between multiply charged cations and singly charged anions. Utilizing ion/ion reactions with fluoranthene has offered a unique method of fragment formation for structural elucidation of biomolecules. Fluoranthene is considered an ideal anion reagent because it selectively causes electron transfer dissociation (ETD) and minimizes PT when interacting with peptides. However, limited investigations have sought to understand how fluoranthene – the primary, commercially available anion reagent – interacts with other biomolecules. Here, we apply deuterium labeling to investigate ion/ion reaction mechanisms between fluoranthene and divalent, metal-adducted carbohydrates (Ca2+, Mg2+, Co2+, and Ni2+). Deuterium labeling of carbohydrates allowed us to observe evidence of hydrogen/deuterium exchange (HDX) occurring after ion/ion dissociation reactions. The extent of deuterium loss is dependent on several factors, including the physical properties of the metal ion and the fragment structure. Based on the deuterium labeling data, we have proposed ETD, PTD, and intermolecular PT – also described as HDX - mechanisms. This research provides a fundamental perspective of ion/ion and ion/molecule reaction mechanisms and illustrates properties that impact ion/ion and ion/molecule reactions for carbohydrates. Together, this could improve the capability to distinguish complex and heterogenous biomolecules, such as carbohydrates. 

Carbohydrates and glycans are integral to many biological processes, including cell-cell recognition and energy storage. However, carbohydrates are often difficult to analyze due to the high degree of isomerism present. One method being developed to distinguish these isomeric species is hydrogen/deuterium exchange-mass spectrometry (HDX-MS). In HDX-MS, carbohydrates are exposed to a deuterated reagent and the functional groups with labile hydrogen atoms, including hydroxyls and amides, exchange with the 1 amu heavier isotope, deuterium. These labels can then be detected by MS, which monitors the mass increase with the addition of D-labels. The observed rate of exchange is dependent on the exchanging functional group, the accessibility of the exchanging functional group, and the presence of hydrogen bonds. Herein, we discuss how HDX has been applied in the solution-phase, gas-phase, and during MS ionization to label carbohydrates and glycans. Additionally, we compare differences in the conformations that are labeled, the labeling timeframes, and applications of each of these methods. Finally, we comment on future opportunities for development and use of HDX-MS to analyze glycans and glycoconjugates. 

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.