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




Makepeace, Karl A.T.

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Proteins 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.



Mass Spectrometry, Structural Proteomics, Crosslinking, Cross-linking, Disulfide bonds, Proteomics, Tau, Mitochondria, Method development, Molecular dynamics, Protein chemistry, Protein crosslinking, Protein cross-linking, Shotgun proteomics, Bottom-up proteomics, Data-dependent acquisition, DDA, Protein-protein interactome, Interactome, Interactomics, Proteinase K, Trypsin, Higher-order crosslinking, Machine learning, Feature engineering, Yeast, Yeast mitochondria, Algorithms, Data analysis, Surface modification, Molecular modelling, Non-specific digest, Structural mass spectrometry, Structural biology, Biochemistry, LC-MS