An Investigation on the Application of Mass Spectrometry Protein Footprinting Technique to Study Interactions of Collagen and Collagen Receptors
Item
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Title
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An Investigation on the Application of Mass Spectrometry Protein Footprinting Technique to Study Interactions of Collagen and Collagen Receptors
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Identifier
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d_2009_2013:469757d9addb:12021
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identifier
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12718
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Creator
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Kirchner, Michele,
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Contributor
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Yujia Xu
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Date
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2013
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biochemistry
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Abstract
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Collagen is one of the most abundant proteins in the human body. There are many different types of collagen; they interact with an assortment of other proteins in the extracellular matrix, and with collagen receptors on the surfaces of cells. In this study, oxidative protein footprinting using Fenton chemistry followed by mass spectrometry was explored as a method to investigate collagen interactions. A known collagen binding protein, the A3 domain of the von Willebrand Factor protein (vWF A3), was chosen as a model. This protein association is the first critical step in blood clotting, and has been the target for the development of new drugs to treat von Willebrand disease, a bleeding disorder. Mass spectrometry protein footprinting could potentially aid in these endeavors. In order to accomplish this objective, original studies using Fenton chemistry labeling of triple helical peptides, and mass spectrometry collagen sequencing studies were carried out. The von Willebrand Factor A3 protein was produced from an Escherichia coli expression system. This work discovered many challenges that have to be overcome for the successful approach of mass spectrometry oxidative protein footprinting by Fenton chemistry to investigate full chain collagen interactions. Additionally, the mix of variable post translational modifications of collagen makes the identification of binding sites by footprinting unreliable. Even sequencing collagen proves problematic because of the many post translational modifications. Despite these setbacks, we did; however, find that the von Willebrand Factor A3 protein binds to a triple helix conformation lacking any of the presumed necessary hydroxyproline sites. We were also able to identify regions in full chain collagen where the oxidation content varied from what was expected, assuming all the prolines in the Y position were hydroxylated.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biochemistry
