Mass Spectrometry Imaging to inform on the pathology of Age-Related Macular Degeneration

Mass Spectrometry Imaging (MSI) has been developed significantly in the past few decades and has been transformed into a technique that not only allows for spatial mapping of analytes of interest but is a key tool in biomarker and medicinal discovery. At Sheffield Hallam University, research using matrix-assisted laser desorption ionisation (MALDI-MSI) has propelled ocular research, oncological research, and many other fields. Additionally, the use of laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP-MSI) has been fruitful for investigations into the ocular pathology of uveal melanoma, and desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) has aided research into certain cancers through biomarker research. These techniques however are seldom combined to create multifaced datasets and had yet to be applied to age-related macular degeneration (AMD) research. Furthermore, the field of ocular pathology has benefitted greatly from decades of extensive immunological research into age-related macular degeneration. More recently, Doyle et al. at Trinity College Dublin have been using immunological techniques to investigate AMD and its pathology, in addition to the wider innate immune system. However, there have been few studies in AMD research that have built upon these works with MS. The work conducted herein offers a platform on which further ocular research can be built. Through utilisation of DESI-MSI for the detection of lipids, MALDI-MSI for the detection of peptides, LA-ICP-MSI for the detection of metals, and micro-particle induced X-ray emission (µPIXE) for the detection of other elements this work has developed a multimodal workflow whereby a rich dataset can be obtained from ocular tissue. The aforementioned immunological techniques have provided great insight into the ocular proteome but reply heavily on antibody specificity. There have been instances where this specificity has been called into question, undermining otherwise reputable research, which has intimated there may be a role for untargeted techniques such as MS in ocular pathology. The development of a high-resolution peptide imaging strategy using tailored sample preparation techniques and employment of modern sample preparation apparatus has provided key initial steps for the discovery of biomarkers of AMD in the ocular proteome (Chapter 2,3). Within this study, employment of this strategy aided the research into the human ocular proteome (Chapter 5) through the tentative identification of NLRP3 peptides within the human retina, which has been subject to ongoing debate as a result of immunological reagent specificity. Literature data has clearly linked the metallome to the onset of AMD, and has recommended supplementation of zinc and copper to protract AMD onset, but there is no clear role for these metals in AMD pathology. With existing studies infrequently using high resolution imaging, and rarely applying quantitative dimensions, there is adequate appeal for clarity on the involvement of essential trace elements in the ocular anatomy. Development and deployment of a semi quantitative imaging strategy for copper and zinc within the ocular anatomy (Chapters 3,4) was able to demonstrate the high spatial resolution imaging capacities achievable using biological tissue by LA-ICP-MSI. Furthermore, the application of this strategy to wildtype and transgenic ocular mouse tissues was able to give an insight into the ocular metallome, and when combined with µPIXE (Chapter 4) could show how trace elements interact with the substructures of the ocular structure in the context of retinal degeneration. With an increasing number of studies utilising untargeted lipidomic data to identify potential biomarkers of disease, there was also scope to include the use of MSI to investigate lipidomics. Using DESI-MSI (Chapter 5) lipid profiles of multiple wildtype and transgenic mouse ocular tissue was characterised and processed using statistical analysis. Additionally, through use of the ‘soft’ ionisation provided by DESI, MALDI-MSI experiments were able to take place in tandem with DESI analysis, allowing 2 datasets to be obtained from a single tissue. With the previous demonstration (Chapter 2) of the efficacy of LA-ICP-MSI on biological tissues previously analysed by MALDI-MSI, this body of work has provided the tools necessary for a 3-technique multimodal approach to imaging of ocular tissue. Consequently, through method development and optimisation for multiple imaging strategies, and the demonstration thereof, this body of work has contributed significantly to the field of ocular pathology by MSI.