Top-Down Mass Spectrometry Characterization of Protein-Ligand Complexes Important to Neurodegenerative Diseases PDF Download

Author: Piriya Wongkongkathep
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Languages : en
Pages : 170

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Book Description
Mass spectrometry (MS) has made significant contributions to protein and proteomics analysis during the past decades from its advantages of speed, sensitivity, specificity, and low sample consumption. While the proteomics field grows rapidly to identify thousands of proteins in a single analysis, "native" mass spectrometry, exploiting the unique features of electrospray ionization (ESI) for delivering large macromolecules to the mass spectrometer, has provided many potential exciting capabilities and applications to structural biology and biochemistry. It can analyze proteins in their native states, i.e., structures present in their native configurations from physiological pH solutions, with minimal sample preparation. In this thesis, I describe the application of native ESI combined with top-down MS using electron capture dissociation (ECD) and ion mobility (IM) to characterize the molecular features of protein-ligand complexes. Binding and structural information can be comprehensively obtained from this experimental platform. Native ESI-MS alone provides molecular mass, stoichiometry, and binding affinity, all from a single analysis. We demonstrate that top-down MS, the fragmentation of intact proteins and protein complexes using MS, offers a powerful capability to elucidate the location of ligand binding on a protein's structure and for probing the surface topology of proteins. Ion mobility mass spectrometry, a recently developed technique that yields information on the structural conformation of molecules, was used to reveal structural changes of proteins upon ligand binding. My thesis focuses on several proteins, including -synuclein (AS), which is a small protein related to Parkinson's disease. AS is natively unfolded at physiological pH, which makes it difficult to study by standard methods such as X-ray crystallography or NMR. Using our mass spectrometry techniques, transition metal binding (copper, cobalt, and manganese) to AS that is associated with accelerating fibril formation was monitored. The binding of a small molecule amyloid inhibitor called molecular tweezer (MT or CLR01) on two model proteins important in neurodegenerative diseases, AS and superoxide dismutase (SOD1), was studied. Tandem mass spectrometry (MS/MS) techniques such as collisionally activated dissociation (CAD) along with ECD were used to characterize the sites of binding of small molecule ligands to proteins. Ion mobility mass spectrometry was implemented to reveal the conformational changes of AS upon metal binding. It was demonstrated that copper can induce the AS protein to collapse into a more compact state, which may provide a hint of the mechanisms behind amyloid fibrillation. Additionally, two new methods to extend the application of top-down MS for protein structure characterization were developed. First, the same molecular tweezer molecule, which has a specificity to bind lysine residues, was used to probe surface residues of proteins. The lysines found to bind to the molecular tweezers identified by top-down MS correlates well with solvent accessibility values, suggesting that the MT compound can be applied as a molecular probe to pinpoint surface active lysine residues. Lastly, supplemental activation methods by ultraviolet and infrared laser irradiation prior to ECD was applied to assist disulfide bond cleavage of complex multiple intermolecular and intramolecular disulfide bond-containing proteins. Backbone bond cleavage from top-down MS was significantly increased when the disulfide bonds were cleaved, allowing more sequence information to be obtained. The new methods described in this thesis extend the applicability of mass spectrometry to provide a more complete picture of a protein's structure.