Rapid deuterium labeling and localization for structural elucidation of carbohydrates.


Carbohydrates are crucial components of life, serving a variety of cellular functions with broad implications. Characterization of carbohydrate structures is a significant step for understanding cell biology. While mass spectrometry (MS) is a powerful tool that provides both qualitative and quantitative information on a number of analytes, ranging in size and functionality, structural characterizations of carbohydrates via MS generally focuses on sequencing rather than analyzing conformations. Hydrogen/deuterium exchange (HDX) is a well-established technique that is traditionally used to study protein conformations and dynamics. However, application of HDX for carbohydrate analysis has been limited due to the rapid HDX rates of carbohydrate hydroxyls, which range in the microsecond timescale. The goal of this work was to develop HDX methods to characterize carbohydrate conformations and provide additional dimension for MS analysis of carbohydrates. HDX was performed in electrospray (ESI) droplets, thus achieving rapid labeling of biologically relevant structures in solution-phase. Residual solvents in ESI sources were found to cause additional deuterium labeling or loss beyond the HDX reactions in bulk-solution (Chapter Two). The HDX in ESI was affected by changes in the equilibrium of solvent vapors inside the source as samples are infused in real-time during analysis. This in-ESI HDX effect affected not only carbohydrates but also rapidly-exchanging functional groups of peptides, indicating broader implications for protein HDX analysis. Chapter Three presents utilization of theta-ESI emitters to achieve variable HDX timepoints of carbohydrates by varying initial ESI droplet sizes from varying ESI tip diameters. Deuterium labels on carbohydrates were localized by collision induced dissociation (CID). Chapter Four investigates scrambling by vibrational activation during CID, concluding that sodium-adduction to carbohydrates reduces scrambling and maintains meaningful HDX information. In Chapter Five, HDX-CID-MS is applied to carbohydrate isomers to assign deuterium labels. Computational methods are employed to examine fragmentation pathways of each carbohydrate based on their conformations and the relative exchange rates of different functional groups. Deuterium localization on carbohydrates, with their limited number of functional groups and narrow range of exchange rates, displays a positive outlook for HDX analysis of carbohydrates that can detect transient inter- and intramolecular interactions of carbohydrates.



Hydrogen/deuterium exchange (HDX). Carbohydrates. Mass spectrometry. Electrospray ionization (ESI).