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Title: Exploring biomolecules, metallodrugs, and their interactions via the use of UHR-FT-ICR mass spectrometry
Author: Wootton, Christopher
ISNI:       0000 0004 6062 4077
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2016
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The work presented herein focuses on the study of novel metallodrugs and their interaction with various possible targets and off-targets in the form of biomolecules such as peptides, proteins, DNA, and small molecules via the use of ultra-high resolution Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry. Beyond traditional platinum(II) metallodrug such as cisplatin and oxaliplatin, new designs of metallodrugs are being conceived to attack and kill cancerous cells via new mechanisms of action in an effort to exceed the potency, selectivity, and effectiveness of metallodrug treatments. These new metallodrugs range from various activation strategies, to specific target binding, and even catalysis of cell-disrupting processes. As a result these novel drugs can have a wide array of effects on various biomolecule species the may encounter in the body. It is the aim of this thesis to show that mass spectrometry, specifically FT-ICR Mass Spectrometry, is uniquely suited to studying the wide array of metal-based drugs, their biomolecule targets/off-targets, and the numerous reaction products produced from their interactions. Though the study of metallodrug-modified biomolecules via mass spectrometry was shown to be challenging in many cases, mass spectrometry is currently the only analytical technique viable for studying the complex systems involved to a useful level of detail. The majority of the thesis focuses on study of isolated biomolecules interacting with novel metallodrugs and the MS and tandem-MS based study of the resulting observable components. A great range of metallodrugs and biomolecule interactions were observed. A photoactivated platinum(IV) metallodrug (trans, trans, trans[Pt(py)2(OH)2(N3)2]) was shown to produce a variety of platinum(II) based modifications to the peptides studied when activated with blue (463nm) visible light, with the ligand configurations varying depending on whether a histidine amino acid residue was present, allowing retention of both pyridine ligands, or not, allowing release of any of the bound ligands. Tandem MS studies using electron based dissociations showed the primary preference of binding to be at the Histidine residues, and when not available the complexes could bind to lysing functional groups, distinctly different behaviour to previously studied Pt(II) complexes. Oxidation of peptide species was also found to be a significant product of these reactions. Tandem MS studies located the oxidation sites to methionine and tryptophan residues, the latter of which provided insights into the oxidation mechanism. The oxidation process was found to be due to a hydroxyl radical process, not a singlet oxygen mechanism. UV/vis and EPR measurements were also undertaken and supported the results found. Studies into the interaction of a potent osmium(III) compound with isolated DNA strands showed that the metallodrug could bind to both guanine and cytosine sites along the biomolecule. Analysis of these species via CAD MS/MS proved challenging due to damage and eventual dissociation of the metal complex modifications. Whereas electron detachment dissociation enabled the elucidation of the two distinct binding locations. CAD MS/MS was found to be useful for studying hydrogen bonded/pi-stacking stabilised structures. Further investigations into DNA MS and MS/MS lead to the study of the platinum(IV) compound interactions with DNA stands, showing a vastly improved rate of reaction for the compound when compared to the previous peptide reactions. In addition uniform retention of pyridine ligands and no oxidation was observed, providing further evidence of the biomolecule playing a key role in the activation process of the Pt(IV) drugs. MS/MS studies of platinated DNA resulted in similar findings to the osmium-based metallodrugs. Experiments with native MS of DNA showed that observation and interrogation of duplex-DNA strands is possible, even with DNA strands which are not stabilised easily in ammonium acetate solutions, and without annealing. MS, MS/MS, and MS3 was achieved on duplex DNA strands using large concentrations of ammonium acetate and potassium chloride solutions, along with extremely carefully tuned MS source and transfer parameters. The effects of metals on tandem-MS techniques was uniquely apparent during the study of functionalised Iridium-based metallodrug modified peptides. The iridium complexes were shown to effectively quench many electrons used during ECD MS/MS. Though the species were still able to be studied using optimised ECD and CAD MS/MS parameters. The reactivity of metal centres was also shown to affect their own bound ligands, as observed herein with rhodium(II) compounds. The rhodium piano-stool complexes were shown to vastly accelerate and enable room temperature activation of C-H bonds within arene ligands towards hydrogen-deuterium exchange experiments, with some compounds achieving full exchange of available groups within just 1 hour. The process was studied using FT-ICR MS to track the exchange process and observe sequential exchange for 10 different rhodium compounds. An iridium analogue was also studied, which was ineffective, displaying the Rh metal centre’s unique chemistry for this reaction. A novel method for the enhancement of the electrospray ionisation process was conceived and developed in order to achieve improvement of analyte charging, so-called “supercharging”. Using a combination of standard electrospray ionisation and atmospheric chemical ionisation, CH5 +“superacid” ions were introduced into the ESI plume and enabled solution-additive free supercharging of analytes under a variety of conditions. The achievement of higher charge states/enhancement of charge is of uniform benefit to biomolecule characterisation and could help reduce the need for solution phase additives which can disrupt many chemical processes and biomolecule structure. The final section of the thesis is concerned with the escalation of metallodrug-biomolecule interaction from isolated biomolecule up to full-cell proteomes via FT-ICR MS. The great array of challenges faced in previous studies is addressed and strategies for accurate and reliable studies of large metallodrug-modified systems are outlined and tested. Two major strategies are proposed, one based on liquid-chromatography mass spectrometry with modified data processing techniques. The other using a niche MS/MS technique known as two-dimensional mass spectrometry, which would enable whole proteome characterisation without chromatographic separation. Preliminary result using both approaches and future outlook are presented.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology ; RM Therapeutics. Pharmacology