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Mass Spectrometry Based Characterization of Protein Structure and Peptide Isomerization

Abstract

Structure is the key factor in protein function. Mass spectrometry has been shown to be a powerful technique for exploring protein structures and post translational modifications. This dissertation describes the application of mass spectrometry based techniques to investigate peptide isomerization and protein solution phase structure.

The first half of the dissertation focuses on the identification of subtle post translational modifications of protein: epimerization and isomerization. The presence of a single D-amino acid in a peptide is very difficult to detect. Mass spectrometry-based approaches rely on differences in fragmentation between peptide epimers. The success of this approach is dependent on the structural sensitivity of the fragmentation method. Radical Directed Dissociation (RDD) is particularly sensitive to the structure of the ion being fragmented. It is demonstrated herein that RDD provides significantly better chiral discrimination than collisional induced dissociation (CID). The combination of RDD and CID is further applied for the identification of peptide epimerization/isomerization in crystallin proteins. The sites that undergo the greatest degree of isomerization in crystallin correspond to disordered regions, which may have important implications for the chaperone functionality of the proteins within the context of aging.

The second half of the dissertation utilizes the ability of 18-crown-6 (18C6) to form noncovalent complexes with cationic groups to examine protein structures in solution. It is demonstrated with model peptides that the 18C6 adduct stability is increased if intramolecular charge complexation is inhibited by steric or competitive binding. Molecular mechanics and dissociation experiments demonstrated that significant structural changes occur upon loss of 18C6 in model peptides. Collisional activation of protein-18C6 complex indicates that lower charge states represent structures that are not similar to gas phase idealized states. Therefore, 18C6 can behave as a pseudo-solvent molecule and preserve protein solution phase structure. 18C6 attachment can also be applied to investigate protein electrostatic surface structure. It is shown that proteins can have completely different surface structures despite the fact that the backbone structures are similar. The results illustrated a new technique to characterize protein surface structure and dynamics.

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