Hyphenated Elemental Mass Spectrometry for the Biosciences

Abstract

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 characterised methods to spatially quantify manganese and its transporter proteins. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) provides a single technological platform to construct quantified images of elements and may be extended to measure biomolecules via incorporation of immunoassays. However, high quality and reproducible analyses require quality assurance across all steps of the workflow including the characterisation of antibodies, nanoparticles and antibody tagging protocols. Accordingly, this thesis introduces a portfolio of methods of hyphenated ICP-MS for quality assurance of elemental and biomolecule analyses.

Chapter 2 introduces novel and universal workflows for the analysis of intact proteins via capillary electrophoresis (CE) and presents guidelines for the targeted selection of appropriate background electrolytes via consideration of the target proteins’ isoelectric point. Neutral dimethyl polysiloxane capillaries with dynamic coatings of cationic cetyltrimethylammonium bromide or anionic sodium dodecyl sulfate, and bare fused silica capillaries were systematically evaluated for the analysis of seven model proteins over a wide pH range. Multiple capillary and background electrolyte combinations were suitable for the analysis of each protein. The concept was demonstrated by the analysis of caseins and whey proteins in milk which separated the most abundant proteins, including the isoforms of A1 and A2 β-casein and β-lactoglobulin A and B.

Chapter 3 presents the development of a simple, robust, and cost-effective interface to hyphenate CE and ICP-MS to enhance the sensitivity and specificity for the analysis of limited volume and complex biological samples. The interface components were thoroughly investigated to highlight crucial aspects that need to be considered when developing and assembling a CE-ICP-MS interface. The interface’s functionality, linearity and robustness were evaluated by separation and quantification of gadolinium-based contrast agents in urine samples collected after magnetic resonance imaging (MRI) examination.

Chapter 4 combined these advancements to determine labelling efficiencies of metal conjugated antibodies by CE-ICP-MS, which are widely used in cytometry and imaging for the identification and examination of protein expression. The number of lanthanide ions per protein was measured in seven MAXPAR™ polymer conjugated antibodies. Variable numbers of lanthanides were observed between different antibodies, as well as antibodies of the same kind, highlighting the importance of quality control workflows. The CE-ICP-MS method was also applied to 15 nm gold nanoparticles to demonstrate feasibility to distinguish unconjugated and antibody conjugated nanoparticles.

Chapter 5 details novel methods of single-particle ICP-MS to characterise the composition, size distribution and particle-particle interactions of (upconversion) nanoparticles. The optimization of ion extraction, ion transport, and the operation of the quadrupole with increased mass bandwidth improved the signal-to-noise ratios significantly and decreased the size detection limits for all nanoparticle dispersions investigated.