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Type: Thesis
Title: Mass Spectrometric Characterisation of Protein Assemblies from Bitis arietans Snake Venom
Author: Bubner, Emily Rose
Issue Date: 2021
School/Discipline: School of Physical Sciences
Abstract: Snake venom is a sophisticated lethal weapon system designed to immobilise and debilitate prey. It is comprised of various bioactive compounds including proteins which contribute to the high potency of venom due to their selectivity when interacting with biological targets. It is believed the interactions that stabilise higher-order protein structures may enhance their activity. A lack of suitable techniques to study dynamic higher-order protein structures, however, has left a significant knowledge gap in understanding how protein interactions and synergistic effects contribute to biological activity and outcomes of envenomation. Thus, an in-depth characterisation of the highly specific mechanisms behind envenomation is lacking at the molecular level. Cross-linking mass spectrometry can be used to study transient and dynamic interactions involved in the function and malfunction of biological systems. Although a well-established analytical method, a lack of commercially available multifunctional cross-linking reagents hampers the diversity of its application. This thesis contributes to new developments in the cross-linking mass spectrometry workflow. A novel cross-linker library has been developed based on a modular cross-linker structure comprised of a reactive group, a linker arm, and an affinity enrichment tag. Derivatives were synthesised by substituting different commercially available hydrocarbons and amino acids. These units can be linked using a small number of simple chemical reactions such as esterification, amide coupling, alkylation and hydrolysis. By incorporating different spacer arm lengths, reactive groups and cleavable groups, derivatives with different functionalities can be synthesised and applied to different protein systems. The utility of this synthetic process was validated through the synthesis of a non-cleavable heterobifunctional linker, a non-cleavable homobifunctional linker and a sulphonium ion cleavable homobifunctional linker. The heterobifunctional derivative contained a non-specific diazirine group and a lysine-specific fluorinated phenyl reactive group along with an azide affinity tag for copper catalysed alkyne azide click enrichment. Both homobifunctional linkers contained two lysine-specific fluorinated phenyl reactive groups and an alkyne affinity tag for enrichment. Lysozyme, which is known to self-associate at neutral pH, was used to develop a cross-linking assay to compare commercially available cross-linker disuccinimidyl sulphoxide (DSSO) with the linkers designed and synthesised in this thesis. The cross-links identified using the novel homobifunctional linkers were consistent with those detected using the commercially available DSSO, suggesting the newly synthesised linkers can provide valuable structural information on complicated protein systems. This thesis also demonstrates the use of an integrated mass spectrometric approach to elucidate the structures of proteins found in the venom of the African Puff Adder, Bitis arietans. Following fractionation of the crude venom, a combination of intact, native and ion-mobility mass spectrometry experiments was performed to study the structure and stoichiometry of the higher-order protein structures found within the venom. Bottom-up proteomic analysis was used to characterise the primary sequence of the protein components and cross-linking mass spectrometry was used to further probe the protein structures proposed by intact and native mass spectrometry. Cross-linking mass spectrometry was also used to stabilise the oligomeric species allowing protein identification. Using this approach, a 120 kDa octameric C-type lectin, a 60 kDa tetrameric C-type lectin and a 30 kDa dimeric C-type lectin were identified as higher-order protein assemblies existing in the venom. Together, the structural information afforded by this work demonstrates the ability to study protein structures in complex biological systems using an integrated mass spectrometry approach. Furthermore, it provides the potential to expand the methods currently available for studying structure-function relationships in other biological assemblies. Following structure elucidation of these venom proteins, functional studies can be performed to explore this relationship in the oligomeric protein species. Not only will this assist in understanding the mechanism of envenomation, but it may also guide the development of venom-based therapeutics and biotechnological tools.
Advisor: Pukala, Tara
Jovcevski, Blagojce
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2021
Keywords: Mass spectrometry
Bitis arietans
cross linking
snake venom
ion-mobility mass spectrometry
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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