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Determining the coordination structure of metalloproteins with mass spectrometry
Due to its versatility, mass spectrometry (MS) has been recognized as an excellent tool for the analysis of peptides and proteins. The structural information that this technique has provided for metalloproteins so far, however, has not extended much to coordination structure. Metal catalyzed oxidation (MCO) reactions in conjunction with MS analysis have been utilized to determine the metal-binding sites of proteins. This method involves first selectively oxidizing the protein by generating reactive oxygen species (ROS) in the immediate vicinity of the metal. These ROS can quickly react with the amino acids that are part of the metal-binding site. The oxidatively modified residues are then identified using a combination of proteolytic enzymes and the peptide sequencing ability of MS. The initial focus of the dissertation was on finding the most specific MCO reaction conditions to site-specifically oxidize the amino acids bound to Cu and Fe in several proteins with known binding sites to test the viability of this approach. Relatively general reaction conditions involving ascorbate as a reducing agent and O2 and/or H2O2 as oxidizing agents have been found. The utility of this MCO/MS approach was assessed by the application of the method to β-2-microglobulin (β2m), which has unknown Cu-binding sites. The MCO/MS approach allowed us to gather information on Cu coordination under conditions that would make such determinations very difficult by other techniques. Interestingly, our results show that different conformers of β2m have different Cu coordination environments, and these results may provide insight into the Cu-induced amyloidogenesis of this protein. The MCO/MS procedure relies on collision-induced dissociation (CID) of oxidized peptide fragments. Hence, a better understanding of the dissociation patterns of oxidatively modified peptides has also been sought to facilitate identification of the oxidized residues. In general, the dissociation patterns of oxidized peptide ions are found to depend on peptide composition, charge state, and type of modification. More specifically, oxidation of methionine and cysteine residues, but not histidine residues, has a dramatic effect on the dissociation of peptide ions when the charge state of the peptide is less than or equal to the number of basic residues in the peptide.
Lim, Jihyeon, "Determining the coordination structure of metalloproteins with mass spectrometry" (2004). Doctoral Dissertations Available from Proquest. AAI3152723.