Browsing by Subject "Electrospray ionization mass spectrometry"
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Item Biopolymer analysis by electrospray ionization and tandem mass spectrometry(2004) Keller, Karin Mia; Brodbelt, Jennifer S.Electrospray ionization was used in conjunction with Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry and quadrupole ion trap (QIT) mass spectrometry to study protein and oligonucleotide ions in vacuo. The results help to identify effective strategies for mass spectral analysis of these macromolecules and provide new insight on their gas-phase behavior. Tandem mass spectrometry experiments were conducted to evaluate different ion activation methods for biopolymer sequencing. Multipole storage-assisted dissociation (MSAD) and sustained off-resonance irradiation collision-activated dissociation (SORI- CAD) were compared for protein analysis in FTICR instrumentation, and infrared multiphoton dissociation (IRMPD) and collisonal activated dissociation (CAD) were compared for oligonucleotide analysis in QIT instrumentation. In both studies, the differences in the observed fragmentation patterns were noted and the underlying reasons for these differences were identified. The relative utility of MSAD vs. SORI-CAD and IRMPD vs. CAD were assessed in terms of their ability to produce diagnostic information that could be used to identify the protein or oligonucleotide under study. Tandem mass spectrometry was also employed to study the dissociation patterns of both DNA/metal and DNA/drug complexes. The preferred fragmentation pathways exhibited by these species were observed to vary with the initial charge state of the precursor. The effect of the oligonucleotide sequence, the identity of the metal ion, and the identity of the drug on these pathways was established and (where possible) interpreted in terms of the specific non-covalent bonding patterns present in the parent complexes. Finally, electrospray ionization was evaluated as a tool for screening molecular recognition in nucleic acid aptamer/small molecule interactions. Gas-phase data for binding stoichiometry and relative binding affinity were compared with the known solution behavior for a series of well-characterized case studies. Any observed discrepancies were rationalized in terms of ligand structure and/or the nature of the intermolecular ligand/aptamer interactions.Item Electrospray ionization tandem mass spectrometric techniques for the analysis of drug/DNA complexes(2007-05) Mazzitelli, Carolyn Leigh, 1979-; Brodbelt, Jennifer S.Many anticancer and antibacterial therapies are based on the interaction of small molecule drugs with DNA. Increasing interest in the development of DNA-interactive agents has fostered the need for sensitive and versatile analytical techniques that are capable of characterizing the DNA/ligand interactions and are compatible with librarybased screening methods. Electrospray ionization mass spectrometry (ESI-MS) has emerged as a useful technique for the analysis of non-covalent complexes formed between DNA and small molecules due to its low sample consumption and fast analysis time. The work presented in this dissertation is aimed at exploring, optimizing, and validating ESI-MS methods for characterizing DNA-ligand interactions. ESI-MS is first used to assess the binding of threading bis-intercalators to duplexes containing different sequences to determine high affinity binding sites of the ligands. Preliminary DNAse footprinting experiments identified possible specific binding sites of the ligands and ESI-MS experiments revealed that the ligands bound to DNA duplexes containing the respective specific binding sequences. The metal-mediated binding of benzoxazole ligands with different side chains to duplex DNA is also examined. Cu²⁺ and Ni⁺ were found to promote the most dramatic increase in ligand binding, and ligands exhibiting the most dramatic metal-mediated or metal-enhanced binding were also determined to be the most cytotoxic. The quadruplex DNA binding selectivity of perylene diimides is evaluated by screening the binding of the ligands to quadruplex, duplex and single strand DNA by ESI-MS. Three ligands, one containing basic side chains, one containing anionic sidechains, and one benzannulated compound were determined to be the most-quadruplex selective. The ESI-MS results correlated well with spectroscopic experiments. The relative gas-phase stabilities of different quadruplex DNA structures were investigated using molecular dynamics simulations and ESI-MS. The stabilities from the E[subscript 1/2] values generally paralleled the RMSD and relative free energies of the quadruplexes based on MD energy analysis. Finally an ESI-MS technique employing the KMnO₄ reaction with DNA to determine conformational changes to the duplex structure upon ligand binding is detailed. Thymines in most intercalator/duplex complexes are more susceptible to oxidation by KMnO₄ than those in duplex DNA. CAD and IRMPD experiments are used to identify the site of oxidation.Item Electrospray ionization tandem mass spectrometry methods for the analysis of DNA and DNA/drug complexes(2010-08) Smith, Suncerae I.; Brodbelt, Jennifer S.; Guziec, Frank S.; Kerwin, Sean M.; Stevenson, Keith J.; Willets, Katherine A.Many anticancer therapies are based on the interaction of small molecule drugs with nucleic acids, particularly DNA. Electrospray ionization tandem mass spectrometry has established itself as an irreplaceable tool for the characterization of DNA adducts produced by alkylating agents, carcinogens, and antitumor drugs, in addition to the characterization of nucleic acid post-transcriptional modifications. ESI-MS was used to assess the non-covalent binding of a novel series of intercalating anthrapyrazoles to duplexes containing different sequences. Relative binding affinities paralleled the shift in melting point of the DNA duplexes measured from a previous study. Upon collisionally induced dissociation of the duplex/anthrapyrazole complexes, different binding strengths were discerned based on the fragmentation patterns. In addition, the interactions of a new series of sulfur-containing acridine ligands, some that functioned as alklyating mustards, with duplex DNA were also evaluated. Non-covalent and covalent binding of each ligand was determined, and the site of adduction (G > A) was revealed for the covalent modifications. The distribution of cross-linked products and mono-adducts by psoralen analogs was also monitored by both LC-UV and IRMPD-MS methods. Reactions at 5’-TA sites were favored over 5’-AT sites. The sites of interstrand cross-linking were determined by fragmentation of the duplex/psoralen complexes by infrared multiphoton dissociation (IRMPD). Ultraviolet photodissociation (UVPD) at 193 nm caused efficient charge reduction of deprotonated oligodeoxynucleotides via electron detachment. Subsequent CID of the charge-reduced oligodeoxynucleotides formed upon electron detachment, in a net process called electron photodetachment dissociation (EPD), resulted in a diverse array of abundant sequence ions which allowed the modification site(s) of three modified oligodeoxynucleotides to be pinpointed to a more specific location than by conventional CID. Electron transfer dissociation (ETD) caused efficient charge reduction of multi-protonated oligonucleotides. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), resulted in rich backbone fragmentation, with a marked decrease in the abundance of base loss ions and internal fragments. ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages, with conservation of weaker non-covalent bonds. In addition, IRMPD and UVPD were used to activate charge-reduced oligonucleotides formed upon electron transfer. ET-IRMPD afforded tunable characterization of the modified DNA and RNA, allowing for modified bases to be directly analyzed. ET-UVPD promoted higher energy backbone fragmentation pathways and created the most diverse MS/MS spectra. The numerous products generated by the hybrid MS/MS techniques (ETcaD, ET-IRMPD, and ET-UVPD) resulted in specific and extensive backbone cleavages which allowed for the modification sites of multiple oligonucleotides to be pinpointed.Item Radiolysis of Amino Acids: A Study Using Raman Spectroscopy, Ultraviolet-Visible Spectrophotometry and Electrospray Ionization Mass Spectrometry(2014-12-17) Lou, JijieAmino acids are basic components of proteins and play a critical role in the development and treatment of chronic diseases. Raman spectroscopy, as a non-destructive tool with little sample preparation, has been widely used in vitro and vivo studies. In the current study, twenty amino acids in solid state and aqueous/0.5 M hydrogen chloride (HCl) solution were previously irradiated to 10 kGy, 25 kGy and 50 kGy by electron beam and analyzed using Raman spectroscopy, ultraviolet-visible (UV-Vis) spectrophotometry and electrospray ionization mass spectrometry (ESI-MS). Raman spectroscopy revealed spectral signatures of radiolysis of amino acids which related to the specific molecular vibration could be used for non-invasive analysis of radiation damage in vivo. Correlation between the absorbed dose and absorbance of amino acids was modeled by exponential saturation to explain the nature of radical production during irradiation. The results from ESI-MS revealed the modification of molecules after radiation. The relative stability to radiolysis of twenty amino acids was estimated from the results of those three techniques and compared with previous work. The ability of three techniques to identify and analyze the effects of radiolysis of amino acids in solution was evaluated. This first attempt to combine these three techniques to explore the radiation effect on amino acids, is promising for the further application in vivo studies.Item Structural characterization and enhanced detection of flavonoids by electrospray ionization mass spectrometry and molecular modeling(2004-08) Zhang, Junmei, 1970; Brodbelt, Jennifer S.Structural characterization and isomer differentiation of flavonoids was investigated by gas-phase hydrogen/deuterium (H/D) exchange and various metal complexation approaches using electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS). Gas-phase H/D exchange was used to probe the conformations, gas-phase acidities, and sites of deprotonation of isomeric flavonoids. The structural factors that promote or prevent H/D exchange were identified and correlated with collisionally activated dissociation (CAD) patterns and/or molecular modeling data. The use of a transition metal and an auxiliary ligand to form flavonoid/transition metal/auxiliary ligand complexes was proved to be more effective than deprotonation for differentiating isomeric flavonoids using chalcones as an example (a subclass of flavonoids). The relative threshold dissociation energies of such transition metal complexes were measured by energy-variable CAD, and the structural features that influence the threshold energies were identified. The conformations, point charges, and helium (collision gas) accessible surface areas obtained by various computational means were used to rationalize the differences in threshold energies of isomeric flavonoids. Aluminum(III) was used to form strong complexes with flavonoids for more effective isomer differentiation of flavonoids. The CAD patterns of the aluminum complexes were used to differentiate flavonoids, and the structures and conformations of representative flavonoids were also identified by high level computational means. Silver complexation was used to conquer the limitation of the transition metal and aluminum complexation (flavonoids must have a 4-keto group and at least one adjacent hydroxyl group). Silver complexation could not only be used for isomer differentiation of individual flavonoids but could be adapted to characterize flavonoids in mixtures coupled with high performance liquid chromatography (HPLC). HPLC-ESI-MS was used as a sensitive method with high selectivity to detect flavonoids in kale and grapefruit juice and metabolites in urine. The major flavonoids in kale were detected and confirmed as quercetin and kaempferol by CAD and isotopic labeling. The growing conditions were found to significantly affect the flavonoid levels in kale. LC/MS and LC/MS/MS were used to identify the individual metabolites and to gain insight into the overall metabolite profiles in human urine after consumption of grapefruit juice.