The Development and Application of Mass Spectrometry-based Structural Proteomic Approaches to Study Protein Structure and Interactions

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dc.contributor.authorMakepeace, Karl A.T.
dc.contributor.supervisorBorchers, Christoph H.
dc.contributor.supervisorHoward, Perry L.
dc.date.accessioned2022-08-26T22:13:58Z
dc.date.available2022-08-26T22:13:58Z
dc.date.copyright2022en_US
dc.date.issued2022-08-26
dc.degree.departmentDepartment of Biochemistry and Microbiologyen_US
dc.degree.levelDoctor of Philosophy Ph.D.en_US
dc.description.abstractProteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions.en_US
dc.description.scholarlevelGraduateen_US
dc.identifier.urihttp://hdl.handle.net/1828/14138
dc.languageEnglisheng
dc.language.isoenen_US
dc.rightsAvailable to the World Wide Weben_US
dc.subjectMass Spectrometryen_US
dc.subjectStructural Proteomicsen_US
dc.subjectCrosslinkingen_US
dc.subjectCross-linkingen_US
dc.subjectDisulfide bondsen_US
dc.subjectProteomicsen_US
dc.subjectTauen_US
dc.subjectMitochondriaen_US
dc.subjectMethod developmenten_US
dc.subjectMolecular dynamicsen_US
dc.subjectProtein chemistryen_US
dc.subjectProtein crosslinkingen_US
dc.subjectProtein cross-linkingen_US
dc.subjectShotgun proteomicsen_US
dc.subjectBottom-up proteomicsen_US
dc.subjectData-dependent acquisitionen_US
dc.subjectDDAen_US
dc.subjectProtein-protein interactomeen_US
dc.subjectInteractomeen_US
dc.subjectInteractomicsen_US
dc.subjectProteinase Ken_US
dc.subjectTrypsinen_US
dc.subjectHigher-order crosslinkingen_US
dc.subjectMachine learningen_US
dc.subjectFeature engineeringen_US
dc.subjectYeasten_US
dc.subjectYeast mitochondriaen_US
dc.subjectAlgorithmsen_US
dc.subjectData analysisen_US
dc.subjectSurface modificationen_US
dc.subjectMolecular modellingen_US
dc.subjectNon-specific digesten_US
dc.subjectStructural mass spectrometryen_US
dc.subjectStructural biologyen_US
dc.subjectBiochemistryen_US
dc.subjectLC-MSen_US
dc.titleThe Development and Application of Mass Spectrometry-based Structural Proteomic Approaches to Study Protein Structure and Interactionsen_US
dc.typeThesisen_US

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