UC Riverside

Protein structure is fundamental for biological functioning. Although there are many safeguards for maintaining protein structural fidelity, proteins are still subject to modifications with deleterious effects. Isomerization of amino acids is one such modification resulting in changes to protein structure and function. Analytical methods to interpret protein structure and locate modifications are required to expand our knowledge of protein structure and its role in biology and disease. Mass spectrometry (MS) based methods are uniquely suited to address these needs. Herein, MS methods are discussed for applications in biomolecular structure studies in several contexts. The development of a method to detect isomerization in human brain tissue is described in Chapter 2. Sample preparation methods, instrument methods, and data acquisition and analysis are described. Using the methods proposed, we located isomerization and post-translational modification (PTM) sites in tau protein and superoxide dismutase, both associated with neurodegenerative disease, in human brain tissue. The methods developed will be used for further investigation of the role of these modifications in Alzheimer’s disease and other neurodegenerative disorders. Next, ultraviolet photodissociation (UVPD) was used to interrogate structural differences induced by isomerization in peptide isomers. Differences in chromophore absorption and ion-molecule reactions were used to differentiate synthetic peptides containing aspartic acid (Asp), glutamic acid (Glu), and serine (Ser) isomers. Molecular modeling was used to propose potential structures in the gas phase. We also performed structure studies in the gas phase on whole proteins. Radical directed dissociation (RDD) and UVPD were used to fragment iodine-modified ubiquitin and myoglobin. Results of these experiments reveal conformational differences between charge states, including high charge states. They also provide information about structural differences and similarities between apo- and holo-myoglobin. Lastly, we analyzed 193 nm UVPD and HCD data to test the statistical confidence of assigning internal fragments. We found that assignments of internal fragments feature a high probability of being assigned when an incorrect sequence is used for assignment. We also report ETD data on an intact protein and peptide to observe differences in terminal ions at varying activation time and propose why complement ions pairs are often not observed in top-down analyses of proteins.