Several innovative tandem mass spectrometric strategies have been developed for the structural determination and isomer differentiation of glycosides and oligosaccharides. Specifically, collisionally activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) are used in conjunction with derivatization methods designed to exploit variations in binding energies or attach chromophores. These include metal complexation incorporating modified neutral auxiliary ligands and covalent derivatization involving site-specific reactions. The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and CAD of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) (flavonoid-H) ligand]⁺ complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. We compare and explore the use of alternative pyridyl ligands, with electron-releasing substituents including 4,7-diphenyl-1,10-phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin and luteolin. A tunable ESI-MS/MS strategy for differentiation of flavone and flavanone diglycoside isomers based on metal complexation with auxiliary ligands that have electron-withdrawing substituents is reported. A series of auxiliary ligands with electronwithdrawing substituents was synthesized in order to tailor the relative metal binding affinities of the ligands and thus directly influence the stabilities, and consequently the dissociation pathways, of the complexes. Upon collisionally activated dissociation, the complexes yield fragmentation patterns in which the abundances of key diagnostic ions are enhanced, thus facilitating isomer differentiation. A strategy for increasing the efficiency of IRMPD in a quadrupole ion trap (QIT) based on another metal complexation strategy is described. Two IR-active ligands (IRALs) that have an IR-active phosphonate functional groups were synthesized. The IR-active groups were therefore incorporated into the analyte complexes via metal complexation. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energyvariable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands. A simplified method for determining the sequence and branching of oligosaccharides using IRMPD in a QIT is described. An IR-active boronic acid (IRABA) reagent was synthesized and subsequently used to derivatize the oligosaccharides prior to IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. The approach is demonstrated for two series of oligosaccharides, the lacto-Nfucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/3635 |
| Date | 29 August 2008 |
| Creators | Pikulski, Michael, 1969- |
| Contributors | Brodbelt, Jennifer S. |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | electronic |
| Rights | Copyright © is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works. |
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