Development of mass spectrometry methods for the diagnosis and stratification of non-alcoholic fatty liver disease

Hughes, James William (2022). Development of mass spectrometry methods for the diagnosis and stratification of non-alcoholic fatty liver disease. University of Birmingham. Ph.D.

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The extraction of intact proteins directly from biological samples for analysis by mass spectrometry shows great promise in the development of both diagnostic and stratification techniques for human disease. Additionally, the ability to identify differentially expressed proteins in disease can provide invaluable information for the development of new therapeutics. In this thesis liquid extraction surface analysis (LESA) and desorption electrospray ionisation (DESI) have been applied to the detection of intact proteins directly from thin tissue sections of human liver. The cohort under analysis suffer from non-alcoholic fatty liver disease (NAFLD), a disease of the liver with increasing health burden throughout the world. Previous genetic studies have suggested that a variant of the liver fatty acid-binding protein (FABP1) is associated with worse disease outcomes. Here, LESA coupled with planar high-field asymmetric waveform ion mobility spectrometry (FAIMS) and high resolution mass spectrometry was applied to identification of FABP1 and its variant in a cohort of 42 human liver samples comprising a range of disease states. The presence of the protein and/or its variant was confirmed by collision induced dissociation or electron transfer dissociation mass spectrometry. A statistically significant differential expression of the variant protein was shown between the disease and control cohorts. In the second part of this work, DESI mass spectrometry imaging (MSI) was applied to 20 samples of human liver tissue, including 15 NAFLD samples and 5 normal samples. These experiments made use of a custom heated capillary and travelling wave ion mobility spectrometry (TWIMS). Intact proteins were successfully detected and their distribution across the tissue visualised. A heterogeneous distribution of proteins was observed in non-alcoholic steatohepatitis (NASH) tissue in which the regenerative nodules and fibrotic tissue were clearly visible. A computational workflow was developed to enhance the speed at which the MSI data be analysed and was successfully demonstrated. In the final part of this work, LESA was combined with a cylindrical FAIMS device and high resolution mass spectrometry with the aim of increasing proteome coverage for intact proteins. Initial work in this area focused on development of a data analysis workflow to estimate the number of intact proteins species across all FAIMS settings and across the entire image using an MSI dataset generated from rat testes, kidney, and brain. Subsequently, LESA FAIMS mass spectrometry images were obtained from human liver tissue samples for three disease stages and the data analysis workflow applied.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Engineering & Physical Sciences
School or Department: School of Chemistry
Funders: Engineering and Physical Sciences Research Council
Subjects: Q Science > QD Chemistry


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