Extending the boundaries of hydrogen/deuterium exchange (HDX) to glycans by establishing the fundamentals of in-electrospray ionization HDX.


The main focus of this dissertation is the development of in-electrospray ionization (ESI) - hydrogen/deuterium exchange (HDX) - mass spectrometry (MS) methodologies to elucidate carbohydrate structures. While HDX-MS is a widely used methodology to study protein structure, dynamics, and protein-ligand interactions, the use of HDX in studying carbohydrate structures has been limited, as carbohydrates contain rapidly exchanging hydroxyl groups which exchange at microsecond to millisecond rates. This rapid rate contrasts with that of the amide groups in proteins that exchange on the order of seconds to days, making traditional in-solution HDX methodologies inapplicable to carbohydrates. To address these limitations, we utilized an in-source HDX methodology to provide a suitable time window to analyze carbohydrates by HDX. The in-source methodology, which was first introduced by the Nikolaev group, involved saturating the ESI source by placing a droplet of D2O on a metal plate. This in-ESI HDX methodology required detailed characterization to address fundamental questions about the structures it detects and the methodology itself. Detailed experimentation and observations of this methodology are presented in Chapters Two and Three. This work establishes several fundamental features of in-ESI HDX experiments that are crucial for designing in-ESI HDX experiments. Firstly, in-ESI HDX samples solvated structures of carbohydrates, as metal-adducted carbohydrates exchange minimally in the gas-phase. Secondly, ESI parameters and changes in solvent affect the number of H/D exchanges of metal-adducted carbohydrates significantly. Thirdly, an internal standard can be utilized to lower the day-to-day variability we observed in in-ESI HDX experiments. Further, characterizing in-ESI HDX of carbohydrate-metal adducts led us to discover that spray-solvent conductivity affects the number of measured H/D exchanges. Changes in conductivity alter the size of ESI droplets, which changes the droplet lifetime, and results in a change in the time available for H/D exchange. Hence, varying conductivity is a method to vary the time for H/D exchange. We have combined the knowledge regarding in-ESI HDX experiments with an internal standard and these conductivity effects to investigate the capabilities of in-ESI HDX to distinguish carbohydrate isomers and probe metal-carbohydrate interactions.



In-electrospray. Hydrogen/deuterium exchange. Carbohydrates. Mass Spectrometry. ESI fundamentals.