Novel methods for the rapid and selective analysis of biological samples using hyphenated ion mobility-mass spectrometry with ambient ionization

The increased use of mass spectrometry in the clinical setting has led to a demand for high sample throughput. Developments such as ultra high performance liquid chromatography and the ambient ionization techniques enable high sample throughput by reducing chromatographic run times or by removing the requirement for sample preparation and fractionation prior to analysis. This thesis assesses the reproducibility and robustness of these high throughput techniques for the analysis of clinical and pharmaceutical samples by ion mobility-mass spectrometry. The rapid quantitative analysis of the urinary biomarkers of chronic obstructive pulmonary disease, desmosine and isodesmosine has been performed by ultra high performance liquid chromatography combined with ion mobility-mass spectrometry. The determination of health status based on the free unbound fraction rather than the total bound and unbound desmosine and isodesmosine, significantly reduces the time taken in sample preparation. The potential for direct analysis of the urinary metabolites from undeveloped TLC plates using a solvent extraction surface sample probe is demonstrated. The use of a solvent gradient for the extraction separates urinary metabolites from salts and other matrix components and allows fractionation of the sample as a result of differential retention on the undeveloped RP-TLC plate. This separation, combined with ion mobility-mass spectrometry provides a rapid ambient ionization method for urinary profiling. The combination of a thermal desorption probe with extractive electrospray ionization has been applied to the direct detection of a known genotoxic impurity from a surrogate active pharmaceutical ingredient. The volatility of the impurity compared to the matrix, allowed selective thermal desorption of the analyte, which was ionized by extractive electrospray and detected by mass spectrometry. The use of a rapid on-probe derivatisation reaction, combined with thermal desorption is demonstrated for the direct determination of urinary creatinine. The aqueous acylation of creatinine significantly increases the volatility of the analyte enabling separation from the urine matrix and analysis by thermal desorption extractive electrospray combined with ion mobility-mass spectrometry

Development of novel mass spectrometric methods for point-of-care mucosal diagnostics

Human mucosal surfaces act as key interfaces between microbiota and host. As such, mucosal sampling using medical swabs is performed for diagnostic purposes that most commonly rely upon subsequent microscopy, culture or molecular-based assays. These approaches are limited in providing information on host response, which is a critical facet of pathology. In this thesis, I sought to test the hypothesis that both presence of specific microbes as well as their interactions with the human host are reflected in the mucosal metabolome and that this information could be exploited for mucosal diagnostic applications. The study aimed to develop a method for rapid, direct metabolic profiling from swabs using desorption electrospray ionisation mass spectrometry (DESI-MS). Method optimisation was conducted to elucidate optimal instrumental and geometrical conditions essential for the swab analysis. The application of the method for mucosal diagnostics was then assessed by characterising the metabolic profile of multiple bodysites (oral, nasal and vaginal mucosa), vaginal mucosa during two different physiological states (non-pregnant vs pregnant) and to detect a pathological state (bacterial vaginosis). Correlation of DESI-MS vaginal metabolic profiles with matched vaginal microbiota composition (VMC) characterised by 16S rRNA-based metataxonomics during pregnancy enabled to robustly predict a Lactobacillus dominant from depleted state but also major vaginal community states types (CST). The predictive performance of DESI-MS based models was comparable to “gold standard” LC-MS based models. Additionally, bacterial metabolite markers predictive of specific microbial genera were identified through matching to a spectral database constructed using pure cultures of commensal and pathogenic microbes often observed in the vaginal microbiome. In summary, DESI-MS has the potential to revolutionise the current way of mucosal based diagnostic by reducing significantly the time-demand needed for the characterisation of VMC, drug or inflammatory response to only few minutes and therefore could enable a faster decision making on patient’s treatment.Open Acces

Biomarkers of mismatch repair deficiency in colorectal cancer and cancer predisposition syndromes

PhD ThesisColorectal cancer (CRC) is the third most common cancer in Western societies and approximately 15% are mismatch repair deficient (MMRd). MMRd CRCs have a distinct prognosis, respond to immunotherapy, and occur at a high rate in patients with Lynch syndrome or constitutional mismatch repair deficiency (CMMRD). Detection of MMR deficiency, therefore, guides treatment and identification of associated cancerpredisposition syndromes. However, there is a need for novel biomarkers to detect MMRd CRC, and innovative assays to improve Lynch syndrome and CMMRD diagnosis. I assessed autoantibodies generated against MMRd CRCs as a liquid-biopsy biomarker for cancer detection, by analysing the sera of 464 Lynch syndrome gene carriers using a recently published, multiplex method. Although autoantibodies correlated with a history of CRC, a lack of signal from patients who developed CRC shortly after sampling suggests the method has poor sensitivity. Microsatellite instability (MSI) is an established biomarker of MMR deficiency. I used single molecule molecular inversion probes to develop a sequencing-based MSI assay with an automated results analysis, suitable as a companion diagnostic for immunotherapy, and for streamlined Lynch syndrome screening. The assay achieved 100% accuracy in 197 CRCs, and was robust to sample variables, including quantity, quality, and tumour cell content. Subsequently, I adapted the MSI assay to detect low-level MSI in non-neoplastic tissues of CMMRD patients. The assay separated all 32 CMMRD patients from 94 controls. For both CRC and CMMRD diagnostics, the MSI assay is cheaper and faster than current methods, and is scalable to large cohorts. These results suggest that the humoral immune response to MMRd CRCs cannot readily be used as a biomarker to detect disease, and that alternatives should be sought. However, the MSI assay could be deployed into clinical practice to meet the high demand for MMR deficiency testing of CRCs and to improve CMMRD diagnostics.the Barbour Foundatio

MSI-based mapping strategies in tumour-heterogeneity

Since the early 2000s, considerable innovations in MS technology and associated gene sequencing systems have enabled the "-omics" revolution. The data collected from multiple omics research can be combined to gain a better understanding of cancer's biological activity. Breast and ovarian cancer are among the most common cancers worldwide in women. Despite significant advances in diagnosis, treatment, and subtype identification, breast cancer remains the world's second leading cause of cancer-related deaths in women, with ovarian cancer ranking fifth. Tumour heterogeneity is a significant hurdle in cancer patient prognosis, response to therapy, and metastasis. As such, heterogeneity is one of the most significant and clinically relevant areas of cancer research nowadays. Metabolic reprogramming is a hallmark of malignancy that has been widely acknowledged in recent literature. Metabolic heterogeneity in tumours poses a challenge in developing therapies that exploit metabolic vulnerabilities. Consequently, it is crucial to approach tumour heterogeneity with an unlabeled yet spatially specific read-out of metabolic and genetic information. The advantage of DESI-MSI technology originates from its untargeted nature, which allows for the investigation of thousands of component distributions, at a micrometre scale, in a single experiment. Most notably, using a DESI-MSI clustering approach could potentially offer novel insights into metabolism, providing a method to characterise metabolically distinct sub-regions and subsequently delineate the underlying genetic drivers through genomic analyses. Hence, in this study, we aim to map the inter-and intra-tumour metabolic heterogeneity in breast and ovarian cancer by integrating multimodal MSI-based mapping strategies, comprising DESI and MALDI, with IMC (Imaging Mass Cytometry) analysis of the tumour section, using CyTOF, and high- throughput genetic characterisation of metabolically-distinct regions by transcriptomics. The multimodal analysis workflow was initially performed using sequential breast cancer Patient-Derived Xenografts (PDX) models and was expanded on primary tumour sections. Moreover, a newly developed DESI-MSI friendly, hydroxypropyl-methylcellulose and polyvinylpyrrolidone (HPMC/PVP) hydrogel-based embedding was successfully established to allow simultaneous preparation and analysis of numerous fresh frozen core-size biopsies in the same Tissue Microarray (TMA) block for the investigation of tumour heterogeneity. Additionally, a single section strategy was combined with DESI-MSI coupled to Laser Capture Microdissection (LCM) application to integrate gene expression analysis and Liquid Chromatography-Mass Spectrometry (LC-MS) on the same tissue segment. The developed single section methodology was then tested with multi-region collected ovarian tumours. DESI-MSI-guided spatial transcriptomics was performed for co-registration of different omics datasets on the same regions of interest (ROIs). This co-registration of various omics could unravel possible interactions between distinct metabolic profiles and specific genetic drivers that can lead to intra-tumour heterogeneity. Linking all these findings from MSI-based or guided various strategies allows for a transition from a qualitative approach to a conceptual understanding of the architecture of multiple molecular networks responsible for cellular metabolism in tumour heterogeneity.Open Acces

Mass spectrometry platforms for higher order structural measurements of protein biotherapeutics Reference procedures for establishing the reproducibility and sensitivity of measurements

Monitoring changes in protein higher order structure (HOS) is an important regulatory requirement for reasons of safety, efficacy and potency of the resultant therapeutic. In particular, as monoclonal antibodies (mAbs) emerge rapidly as a dominant class of therapeutics, so does the need for suitable analytical technologies to monitor for changes in HOS of these complex large biomolecules. Reference materials (RM) serve a key analytical purpose of benchmarking the suitability and robustness of analytical procedures for both drug producers and regulators. Here two different model systems, of increasing complexity based on commercially available RMs, have been developed and used to illustrate the assessment of repeatability and reproducibility of a range of mass spectrometry based analytical platforms for protein HOS measurements. The simplest model protein RM, recombinant human growth hormone, rhGH, was used to illustrate the development and systematic evaluation of more established platforms hydrogen deuterium exchange-mass spectrometry (HDX-MS) and ion mobility spectrometry-mass spectrometry (IMS-MS) and a more novel platform, fast photochemical oxidation of proteins (FPOP-MS) for protein HOS measurements. Both global and localised changes in rhGH HOS, induced due to the presence of zinc ligands were identified using the methods developed and cross-platform measurements compared. Structural conclusions were underpinned using a statistical approach developed for HDX-MS measurements in which measurement variability was differentiated from small but significant changes in HOS. These HDX-MS and IMS-MS methods approaches were validated using a mAb-based RM from the National Institute of Science and Technology (NISTmAb) and two Fc-glycan variants generated using a simple enzymatic protocol and structural changes characterised. Measurements reproducibly demonstrated decreases in structural stability as a result of loss of Fc-glycan structure. These data promote the use of these, rhGH and NISTmAb RM based model systems, for both validating and establishing the sensitivity of analytical methods for the detection of HOS changes of mAbs and other protein therapeutics. These data also demonstrate the suitability of HDX-MS, IMS-MS and FPOP-MS as valuable analytical methods within the biopharma toolbox

The design and Synthesis of peptide-inspired antileishmanial agents

Leishmaniasis is a tropical disease caused by protozoan parasite of the genus Leishmania. The temporins are a class of antimicrobial peptides (AMPs) and have documented antibacterial and antileishmanial activity. Temporins A, B, C, F, L and 1Sa were synthesised. Fluorescein and tetramethylrhodamine, used as biological imaging agents, were attached to temporins A and B and used in biological testing to track the progress of the peptides through infected macrophage cells, and in an in vitro skin model. Temporins A and L were found to be active against both promastigotes and amastigotes, and alanine and lysine scans of these peptides were performed to attempt to identify any residues causing activity. No residues to this effect were identified, however based on this work, the largest library of antimicrobial peptides to date was synthesised and tested gainst Leishmania mexicana promastigotes and axenic amastigotes. Data obtained was subsequently used in the first reported study of computational modelling to predict the sequences of antileishmanials peptides. Based on this work, peptide sequences were predicted that may show activity as antileishmanials agents. The Ciliatamides consist of three lipopeptides named Ciliatamides A-C, of which Ciliatamide B was shown to possess high levels of antileishmanial activity. (S,S), (R,S), (S,R) and (R,R) forms of Ciliatamide B were synthesised and used in biological testing. The activity of both temporins A and B was assayed against L. mexicana promastigote and axenic amastigotes, as well as murine macrophages: Allowing for an as yet undocumented comparison between both stages of the Leishmania spp. lifecyle. Differences were noted in the activity of the promastigotes and amastigotes lifecycle stages of the parasite, with promastigotes being significantly more responsive to AMPs than the amastigotes. As all previous studies had taken place on the promastigotes lifecycle stage, this finding must be taken into consideration in planning future studies

Development of proteomic techniques for biomarker discovery

The main aim of my research, presented here, is to develop proteomic research techniques, for their use in biomarker discovery and identification. This is broken down into three main chapters: • Biomarker Identification in Stroke Brain guided by MALDI-imaging. • Evaluation of the Effectiveness of Heat Treatment for Prevention of Proteomic Sample Degradation using Label Free Relative Quantitation. • Discovery and Identification of Biomarkers for Hypertension Within these chapters special attention is paid to sample preparation, development and assessment of new methods for biomarkers discovery and identification, the importance of experimental design and the application of relevant and useful statistical methods to enable the mining of useful information from rich datasets. The biological changes in stroke induced mouse brain tissue are studied, the prevention of degradation to tissue samples by a novel heat treatment method and changes to plasma samples from hypertensive, wild type and a congenic strain of rat are also studied. The outcomes of this work are multiple, namely: • The identification of a possible marker for stroke from mouse brain tissue • The effect of a new heat treatment device on proteomic data obtained from mouse brain tissue • The novel application of a statistical analysis to a new type of dataset (LC-MS and label free quantitation of biological samples) • The identification of possible biomarkers for hypertension in rat plasma using this metho