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    Improving the Reliability of Peptide-Zinc (II) Binding Constant Determination with Mass Spectrometry

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    Author
    Parrish, Whitney
    Abstract
    Electrospray Ionization Mass Spectrometry (ESI-MS) has become a powerful technique for analyzing protein- and peptide-ligand interactions and quantifying their binding constants (K[subscript]b). However, ESI-MS as an analytical method has several drawbacks, including one particular problem with the analysis of zinc (II)-containing complexes. During sample infusion through a stainless steel ESI emitter, zinc (II) can be deposited onto the emitter. Therefore, what is being detected is not indicative of what is really present in solution and K[subscript]b will be falsely low. Additionally, ligands present in solution may nonspecifically bind to the species of interest during ESI. This nonspecific binding causes K[subscript]b values to be falsely high. Lastly, in-source (gas-phase) dissociation can also occur, meaning the peptide-ligand complex can partially or completely dissociate during ESI. This dissociation causes K[subscript]b values to be incorrectly low. Each of these problems has been addressed individually, but the goal of this project is to address all three problems in one set of experiments to obtain a more reliable binding constant for a model system. For this work, the model system that was chosen to study was the [beta]-amyloid peptide with zinc (II), in particular the 1-16 and 1-28 fragments. Reference methods have been developed by the Klassen group to correct for nonspecific binding and in-source dissociation. The reference peptide method adds a reference peptide that does not bind the peptide or ligand of interest to solution to account for any nonspecific binding. In our experiments, neurotensin was used as the reference peptide. The reference ligand method adds a reference ligand that does bind the peptide of interest to solution to account for any in-source dissociation that may be occurring. Copper (II) was used as the reference ligand because it binds the [beta]-amyloid peptide with a high affinity that has been previously measured and reported by many researchers. The emitter material was changed from stainless steel to glass, and we have shown that this material change prevents zinc (II) deposition from occurring. Initial data was obtained showing appropriate binding percentages in a 1:1 [beta]-amyloid-zinc (II) solution based on equilibrium calculations. Optimized source conditions were determined based on the binding percentages obtained. A titration style experiment was designed to determine the binding constant for both model systems. The K[subscript]b value obtained for [beta]-amyloid (1-16):Zn²⁺ complex was 1.8 (± 0.2) x 10⁸ M⁻¹. Two K[subscript]b values were determined for the [beta]-amyloid (1-28):Zn²⁺ complex. These two values are 6.7 (± 0.4) x 10⁴ M⁻¹ and 3.2 (± 0.2) x 10⁸ M⁻¹. Even though this work was successful in determining a binding constant for a peptide-zinc (II) system using ESI-MS, there is variability in the results obtained. The common feature of MS and other techniques that are used to determine K[subscript]b values is reproducibility. This illustrates the need to combine complementary techniques when studying peptide-ligand systems and quantifying their binding constants.
    URI
    http://hdl.handle.net/10342/4006
    Subject
     Chemistry, Analytical; Analytical chemistry 
    Date
    2012
    Citation:
    APA:
    Parrish, Whitney. (January 2012). Improving the Reliability of Peptide-Zinc (II) Binding Constant Determination with Mass Spectrometry (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/4006.)

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    MLA:
    Parrish, Whitney. Improving the Reliability of Peptide-Zinc (II) Binding Constant Determination with Mass Spectrometry. Master's Thesis. East Carolina University, January 2012. The Scholarship. http://hdl.handle.net/10342/4006. April 19, 2021.
    Chicago:
    Parrish, Whitney, “Improving the Reliability of Peptide-Zinc (II) Binding Constant Determination with Mass Spectrometry” (Master's Thesis., East Carolina University, January 2012).
    AMA:
    Parrish, Whitney. Improving the Reliability of Peptide-Zinc (II) Binding Constant Determination with Mass Spectrometry [Master's Thesis]. Greenville, NC: East Carolina University; January 2012.
    Collections
    • Chemistry
    • Master's Theses
    Publisher
    East Carolina University

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