Hyphenated Elemental Mass Spectrometry for the Biosciences
- Publication Type:
- Thesis
- Issue Date:
- 2022
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The underlying biological mechanisms of widespread radioresistance of many human tumours remain elusive despite decades of investigations. Research efforts have largely focussed on the genomics/proteomics-based enzymology of DNA repair and free radical scavenging enzymes such as the superoxide dismutases. A recent novel hypothesis is that radiation resistance is predominantly underpinned by non-enzymatic complexes of manganese and small molecular metabolites. These complexes are thought to act as free radical scavengers which provide metabolic radioprotection that render cells variably resistant to the products of ionising radiation.
Multiple influx and efflux metal transporters are involved in manganese homeostasis and are potentially differentially expressed on the surface of cancer cells, leading to variable concentrations of manganese within tumours. Uncovering the mechanisms of tumour radioresistance requires complementary, reliable, and well characterized methods to spatially quantify manganese and its transporter proteins. Accordingly, this thesis introduces a portfolio of methods of hyphenated inductively coupled plasma-mass spectrometry (ICP-MS) for quality assurance of elemental and biomolecule analyses.
The thesis first establishes universal workflows for the analysis of intact proteins by capillary electrophoresis (CE) and formalises guidelines for the targeted selection of appropriate background electrolytes via consideration of the proteins’ isoelectric point. Secondly, a simple and inexpensive interface to hyphenate CE and ICP-MS is presented to enhance the sensitivity and specificity for the analysis of low volume and complex biological samples. Thirdly, the CE-ICP-MS interface was used to characterize the labelling efficiencies of metal conjugated antibodies. A workflow to determine the number of lanthanide ions per protein in MAXPAR™ polymer conjugated antibodies was developed, and the method was further applied to distinguish between un-conjugated and antibody-conjugated gold nanoparticles. Novel methods based on single-particle ICP-MS are introduced in the following chapter to characterize the composition, size distribution, and particle-particle interactions of gold nanoparticles and lanthanide doped upconversion nanoparticles. The value of the developed techniques and their potential deployment was lastly demonstrated with the immuno-mass spectrometry imaging and elemental bioimaging of manganese transporters and transition metals in human melanomas.
The methods presented in the thesis will assist future investigations of the role of manganese and its transporter protein for tumour radioresistance and are applicable to the study of the function of many biological processes which often rely on the up- and down regulation of proteins, and the interaction of metals with biomolecules.
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