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    Site-specific analysis of glycosylated proteins using mass spectrometry

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    umi-ku-2339_1.pdf (2.579Mb)
    Issue Date
    2008-01-16
    Author
    Irungu, Janet W.
    Publisher
    University of Kansas
    Format
    181 pages
    Type
    Dissertation
    Degree Level
    PH.D.
    Discipline
    Chemistry
    Rights
    This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
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    Abstract
    Among the numerous post-translational modifications that a protein can undergo, glycosylation is by far the most common, having the most profound influence on the structural and functional properties of the protein. Therefore, profiling glycosylation patterns in glycosylated proteins and defining the structures and locations of these glycans is important in understanding the structure-function relationship of glycans in glycosylated proteins. The work presented herein focuses on applying different mass-spectrometric methods to profile glycosylation patterns in glycoprotein hormones and HIV envelope proteins. To determine the structures and locations of the glycans on these proteins, a glycopeptide-based mass mapping approach was employed. Glycoprotein hormones mainly contain acidic glycans that are highly sulfated and/or sialylated. These acidic functional groups affect the biological clearance of these proteins. To characterize the glycan structures on glycoprotein hormones, we used a non-specific enzyme to generate small glycopeptides that are easier to separate and analyze. However, analysis of these glycopeptides can be challenging since it involves simultaneous analysis of two unknowns; the peptide and the glycan portions. To facilitate identification of the peptide portion, we developed a web-based tool (GlycoPep ID) that utilizes a characteristic product ion observed in (-)MS/MS data of these glycopeptides. To identify the glycan portion, since (-) MS/MS analysis gives very minimal glycan structural information; we developed an ion-pairing approach, which provides a wealth of structural information on the glycan portion of these glycopeptides. Finally, an HIV envelope protein, CON-S gp140∆CFI, a potential vaccine candidate for HIV/AIDS, was characterized. This protein is extensively glycosylated with over 50% of its mass constituting of glycans. Although these glycans play a major role in viral defense mechanism against the host immune system, the structures and locations of these glycans are still not yet known. To develop an efficacious vaccine against this virus, a complete characterization of these glycans is required. A full glycosylation site-specific analysis of glycans in this protein was performed. This information provided biological insights into why CON-S gp140∆CFI is a good immunogen, thus a potential candidate for an HIV vaccine.
    URI
    http://hdl.handle.net/1808/4370
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    • Dissertations [4473]
    • Chemistry Dissertations and Theses [336]

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    785-864-8983
    KU Libraries
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    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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