Spatial Cholesterol Metabolism: A Mass Spectrometer for Better Diagnosis and Understanding of Disease

Disturbed cholesterol metabolism (including its synthesis and transport) is linked to many disease states including neurodegenerative, neurodevelopmental and metal health disorders, metabolic disease and microbial infections. In addition, many rare diseases show features of disturbed cholesterol metabolism. Research in many groups in the UK embraces cholesterol metabolism, but there are few laboratories which specifically focus on the analysis of the myriad of intermediates involved in pathways transforming cholesterol to bile acids and steroids. The Biomedical Mass Spectrometry Laboratory in Swansea University Medical School is one such laboratory and with new mass spectrometry (MS) equipment from the MRC Mid-Range Equipment Fund will provide a resource for researcher in the UK to access specialist MS equipment, expert knowledge in sample preparation, data analysis and its interpretation.

MS represents the primary technology in the study of cholesterol metabolism (cholesterolomics) and when combined with liquid chromatography (LC) is suitable for the unbiased identification and quantification of the greatest number of metabolites in a complex mixture. Security of metabolite identification is provided by orthogonal data including retention time, accurate mass measurement, and fragmentation patterns, preferably, in the case of cholesterol metabolites, multistage fragmentation (MSn). A recent addition to MS-tool box is MS-imaging (MSI) adding further to the available bioimaging technologies.

The current proposal is for a high-resolution MS instrument with MSn and UVPD capabilities which can be combined with existing interfaces for LC-MS, high spatial-resolution AP-MALDI-MSI and high sensitivity LESAplus-LC-MSI already in Swansea. The AP-MALDI source offers <10 micrometer spatial resolution while LESAplus-LC-MSI provides isomer separation and with pg/mg sensitivity. The instrument will be a resource for MRC-remit research in the UK. The new instrument will underpin scientific research in several key strategic priority areas for MRC which involve cholesterol metabolism, including (1) neuroscience and mental health; (2) infection and immunity; (3) molecular medicine; (4) clinical and translational research; and (5) global health.

There is considerable research interest in the involvement of cholesterol metabolism (including its synthesis and transport) in differing disease states. One way to learn more about such diseases is to monitor how cholesterol metabolism differs between patients and healthy people (and during disease progression) or between animal/cell models and wild type. This can be achieved by mass spectrometry analysis of fluids and tissue sample. By using mass spectrometry imaging (MSI) defined metabolites can be quantitatively visualised from tissue slices, revealing the metabolic differences between e.g. lesions and healthy tissue. Provision of a new mass spectrometer at Swansea, which is an acknowledged centre for the analysis of the cholesterol metabolic pathway, having developed EADSA technology (US patent US9851368B2), will provide a UK resource for biomedical scientists whose research requires an investigation of cholesterol metabolism.

The new instrument will be capable of fluid analysis and with already existing equipment in Swansea, mass spectrometry imaging (MSI). To maximize the security of metabolite identification the instrument will be able to record high-resolution (500,000) accurate mass (<1ppm) spectra and will be able to generate MSn fragmentation trees using different modes of fragmentation, i.e. quadrupole-like, ion-trap-like and UVPD. We have found MSn to be particularly important for the structural characterisation of isomeric oxysterols and bile acid precursors, while UVPD has been shown to be able to locate double bond positions in sterol esters. The instrument will support research in the MRC strategic priority areas including neuroscience and mental health; infection and immunity; molecular medicine; clinical and translational research; and global health. The results generated will help define biomarker panels for the definition and monitoring of the progression of disease and assist in the development of therapy.