Loughborough University
Thesis-2014-Camp.pdf (5.43 MB)

Combining elemental and molecular mass spectrometry to study 3 types of biologically important compounds: DNA, phosphopeptides and anticancer drugs

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posted on 2014-07-07, 07:48 authored by Claire L. Camp
Mass spectrometry was used to investigate three important biological molecules, deoxyribonucleic acid (DNA), phosphopeptides and oxaliplatin. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The method developed quantified phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all 4 DNA mono-nucleotides and 5 UMP. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). Protein phosphorylation and de-phosphorylation is one of the most common signalling pathways within cells, it is involved in regulating cellular processes, mediating enzyme inhibition, protein-protein recognition and protein degradation. A novel approach to the selective detection of phosphopeptides based on the incorporation of a metal tag, gallium N,N-biscarboxymethyl lysine (Ga-LysNTA), in solution before separation and detection by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS) was developed. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of the gallium tag with platelet derived growth factor beta receptor (β-PDGF), a small phosphopeptide. In addition molecular modelling was used to investigate the energetically favoured structures of both the Ga-LysNTA material and the β-PDGF-Ga-LysNTA complex. The complexation of the Pt-based anti-cancer drug oxaliplatin (OxPt) with biological ligands other than DNA is believed to be a major cellular sink for the drug reducing its therapeutic potential and acting as a potential cause of toxicity. The role of the naturally abundant cytoplasmic dipeptide ligand β-alanyl-L-histidine dipeptide (carnosine) in OxPt detoxification was investigated. Various mass spectrometry techniques employing electrospray ionization and chip nanospray were employed to study the interaction of oxaliplatin with carnosine as well as two of its derivatives β-alanyl-N-methylhistidine (anserine) and N-acetylcarnosine (NAC). Evidence of complexation between OxPt and each of the three ligands examined is presented. Most species observed were unambiguously assigned and compared to their theoretical isotopic patterns.





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© Claire Louise Camp

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A dissertation thesis submitted in partial fulfilment of the requirement for the award of the degree Doctor of Engineering (EngD), at Loughborough University.


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Qualification name

  • EngD

Qualification level

  • Doctoral

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