Development of Novel Tandem Mass Spectrometric Strategies towards LC/FT-ICR MS-Based Structural Determination in Metabolomics and Peptidomic Analysis.

Abstract

Mass spectrometry (MS)-based metabolomics offers quantitative analyses with high selectivity and sensitivity, and the potential to identify metabolites. MS is often combined with separation techniques such as liquid chromatography (LC). Metabolic profiling of Jurkat T cells was performed with hydrophilic interaction liquid chromatography (HILIC)/Fourier transform ion cyclotron resonance (FT-ICR) MS in collaboration with Dr. Opipari’s group in the Medical School, University of Michigan. The developed HILIC/FT-ICR MS could detect metabolic changes of T-cells following metabolic perturbation. However, many of the observed metabolites could not be identified using currently available metabolic databases, which reflects a major challenge encountered in mass spectrometric metabolite analysis. Currently, mass spectral libraries are not diverse enough to allow identification of the hundreds to thousands of detected metabolites. FT-ICR MS can provide improved metabolite identification due to its accurate mass capabilities and alternative MS/MS fragmentation techniques, including infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD). These techniques can often provide complementary structural information compared to collision induced dissociation (CID) for various kinds of biomolecules. However, they have seen very limited, if any, application to metabolite characterization. Thus, most of the work in this thesis was devoted to method development for structural characterization and identification of small organic molecules including metabolites and peptides. Alternative strategies available in FT-ICR MS were successfully applied to provide structural information of phosphate-containing metabolites, fatty acids, and phospholipids with and without metal adduction. In addition, it is shown, for the first time, that negatively charged peptide ions can capture electrons, resulting in unique radical species, which dissociate via peptide backbone bond cleavages while retaining posttranslational modifications. Other work includes phosphate-containing metabolite enrichment with TiO2 micro-tips to overcome ion suppression caused by highly abundant polar metabolites, and structural determination of lipopolysaccharide (LPS) using IRMPD.