Advanced applications of mass spectrometry for isomer differentiation and analysis of biomolecules

The work presented herein focuses on the implementation of advanced fragmentation techniques with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to distinguish between isomeric species, including small metabolites and peptides. Applications of matrix-assisted laser desorption ionisation- time of flight mass spectrometry (MALDI-TOF MS) and FT-ICR MS for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are also investigated in this thesis. The differentiation and relative quantification of isomeric species is of importance as the subtle changes in their physical structures may significantly impact their biological function. Current studies have demonstrated the potential of applying tandem mass spectrometry (MS/MS) techniques for direct isomer characterisation via generation of diagnostic fragment ions. Thus, the application of MS/MS methods has been explored in this thesis to characterize and relatively quantify the isomeric products of deamidation (chapter 2), modified tau and pi N-methylated actin peptides (chapter 3), and dihydroxylated vitamin D3 isomers (chapter 4). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus, responsible for causing coronavirus disease 2019 (COVID-19). In this work, MALDI-TOF MS was primarily used for the optimisation experiments to detect SARS-CoV-2 biomarker proteins including the nucleocapsid (N-protein) and the spike glycoprotein (S-protein). Focus was placed on various viral protein enrichment and extraction methods, which were applied to the standard SARS-CoV-2 proteins and then to COVID-19 negative and positive patient swab samples. The final chapter of this thesis provides a conclusion on all the results presented herein and provides an outlook for future research. This can be used to further develop the current experimental work on the use of MS/MS techniques for the differentiation and relative quantification of various isomeric compounds as well the improvement of viral protein enrichment methods for MALDI-TOF MS analysis of SARS-CoV-2 biomarker proteins in complex patient samples

Enhancing Biomolecule Analysis and 2DMS Experiments by Implementation of (Activated Ion) 193 nm UVPD on a FT-ICR Mass Spectrometer

Ultraviolet photodissociation is a fast, photon-mediated fragmentation method that yields high sequence coverage and informative cleavages of biomolecules. In this work, 193 nm UVPD was coupled with a 12 Tesla FT-ICR mass spectrometer and 10.6 μm infrared multi-photon dissociation to provide gentle slow-heating of UV-irradiated ions. No internal instrument hardware modifications were required. Adjusting the timing of laser pulses to the ion motion within the ICR cell provided consistent fragmentation yield shot-to-shot and may also be used to monitor ion positions within the ICR cell. Single-pulse UVPD of the native-like 5+ charge state of ubiquitin resulted in 86.6% cleavage coverage. Additionally, IR activation post UVPD doubled the overall fragmentation yield and boosted the intensity of UVPD-specific x-type fragments up to 4-fold. This increased yield effect was also observed for the 6+ charge state of ubiquitin, albeit less pronounced. This indicates that gentle slow-heating serves to sever tethered fragments originating from non-covalently linked compact structures and makes activation post UVPD an attractive option to boost fragmentation efficiency for top-down studies. Lastly, UVPD was implemented and optimized as a fragmentation method for 2DMS, a data-independent acquisition method. UVPD-2DMS was demonstrated to be a viable method using BSA digest peptides as a model system

Multimodal tandem mass spectrometry techniques for the analysis of phosphopeptides

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging

Differentiation of dihydroxylated vitamin D3 isomers using tandem mass spectrometry

Vitamin D compounds are a group of secosteroids derived from cholesterol that are vital for maintaining bone health in humans. Recent studies have shown extraskeletal effects of vitamin D, involving vitamin D metabolites such as the dihydroxylated vitamin D3 compounds 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Differentiation and characterization of these isomers by mass spectrometry can be challenging due to the zero-mass difference and minor structural differences between them. The isomers usually require separation by liquid chromatography (LC) prior to mass spectrometry, which adds extra complexity to the analysis. Herein, we investigated and revisited the use of fragmentation methods such as collisional induced dissociation (CID), infrared multiphoton dissociation (IRMPD), electron induced dissociation (EID), and ultraviolet photodissociation (UVPD), available on a 12T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) to generate characteristic fragments for the dihydroxylated vitamin D3 isomers that can be used to distinguish between them. Isomer-specific fragments were observed for the 1,25-dihydroxyvitamin D3, which were clearly absent in the 24,25-dihydroxyvitamin D3 MS/MS spectra using all fragmentation methods mentioned above. The fragments generated due to cleavage of the C-6/C-7 bond in the 1,25-dihydroxyvitamin D3 compound demonstrate that the fragile OH groups were retained during fragmentation, thus enabling differentiation between the two dihydroxylated vitamin D3 isomers without the need for prior chromatographic separation or derivatization