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  • Determination of site-specific glycan heterogeneity on glycoproteins

    Author(s)
    Kolarich, Daniel
    Jensen, Pia H
    Altmann, Friedrich
    Packer, Nicolle H
    Griffith University Author(s)
    Kolarich, Daniel
    Packer, Nicki
    Year published
    2012
    Metadata
    Show full item record
    Abstract
    The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by ...
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    The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3–5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.
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    Journal Title
    Nature Protocols
    Volume
    7
    Issue
    7
    DOI
    https://doi.org/10.1038/nprot.2012.062
    Subject
    Chemical sciences
    Biological sciences
    Biochemistry and cell biology not elsewhere classified
    Biomedical and clinical sciences
    Publication URI
    http://hdl.handle.net/10072/336566
    Collection
    • Journal articles

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