Improving Characterization of Small Molecules using Enhanced Molecular Reaction and Ionization Techniques
Date of Graduation
Eberly College of Arts and Sciences
Stephen J. Valentine
Glen P. Jackson
John B. Mertz
Metabolomics is an emerging “omics” field, which is comprised of the study of small molecule metabolites in biological systems. It can be argued that the abundances of metabolites within biological samples, such as tissue and plasma, are indicative of various physiological states. Thus, metabolomics analysis has the potential in the clinical scientific field to be an effective tool for early disease diagnosis as well as for proposing and monitoring new treatments for diseases. However, full characterization of the metabolome complement is often hampered by the wide diversity of metabolites, which also exhibit an extremely large concentration range in biological samples. Added to these challenges is the presence of numerous isomeric and isobaric species thereby presenting difficulties for analytical platforms.
Hybrid techniques such as liquid chromatography (LC) and gas chromatography (GC), combined with mass spectrometry (MS) have been widely used in complex mixture analysis associated with metabolomics investigations. For example, LC-MS ‘omics methods have been broadly utilized to investigate conditions such as cardiovascular disease, cancer, diabetes and neurodegenerative disorders. Despite progress in techniques for metabolome characterization, the major challenge of accurate compound identification remains and stems primarily from an inability to distinguish isomeric and isobaric species by MS as well as to identify the low-abundance species within metabolomics mixtures.
The research presented here focused on developing novel techniques combined with mass spectrometry to significantly enhance the ability to accurately identify challenging metabolites. In one embodiment, rapid, solution-phase hydrogen deuterium exchange (HDX) coupled with MS was demonstrated as a means for distinguishing small-molecule metabolites. Additionally, in this work, a new approach for predicting the solution-phase hydrogen/deuterium exchange (HDX) reactivity for new compounds (e.g., newly emerging drugs) using hydrogen-type parameterization was demonstrated. In another study, experiments attempted to address the extremely challenging structural diversity of carbohydrate compounds including isomeric species. The power of using a combination of orthogonal analytical measurements for distinguishing monosaccharide stereoisomers as well as disaccharide linkage isomers by employing rapid (μs timescale) solution-phase HDX with MS detection.
In one noteworthy project, field-enabled cVSSI has been combined with high-flow, liquid-chromatography (LC)-MS to establish the current ionization advantages for metabolomics investigations. This work was directed towards developing capabilities for identifying lower-abundance species that may be masked by signals from higher-abundance species in a complex mixture. In the final installment of this work, a combination of techniques, ion mobility spectrometry-mass spectrometry (IMS-MS), and gas-phase HDX using D2O vapor were used as tools to distinguish and establish unique isotopic distribution patterns for disaccharide ion species. Molecular dynamics (MD) simulations were applied to in-silico structures for HDX accessibility modeling. The results suggest that different charge assignments for different ions may be in part responsible for the different HDX levels observed. The work described in these four projects, is discussed with an eye toward future efforts to enable the wide-scale adoption of these methods in metabolomics workflows.
Majuta, Sandra N., "Improving Characterization of Small Molecules using Enhanced Molecular Reaction and Ionization Techniques" (2021). Graduate Theses, Dissertations, and Problem Reports. 8113.