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  • Removal of N-Linked & O-Linked Glycans from Glycoproteins

    Removal of oligosaccharides from glycoproteins, termed deglycosylation, is often used in order to simplify analysis of the peptide and/or glycan portion of a glycoprotein. Detailed knowledge of the glycan structures helps to correlate them to their respective function. To do this, tools are required for highly sensitive analysis of glycan chains. Both chemical and enzymatic methods exist for removing oligosaccharides from glycoproteins. However, chemical methods such as β-elimination with mild alkali (1) or mild hydrazinolysis (2) often results in the degradation of the protein; whereas, enzymatic methods such as treating with PNGase F (NEB #P0704, NEB #P0705) or Endo H (NEB #P0702, NEB #P0703) (3) for N-glycans are much gentler and can provide complete sugar removal with no protein degradation. Unfortunately, there are no general endoglycosidases that can give complete removal of all O-linked glycans. The broadest specificity O-glycosidase (NEB #P0733, NEB #E0540) is able to cleave only core 1 and core 3 O-glycans (4). Using the O-glycosidase in combination with an appropriate mix of exoglycosidases allows the removal of large O-glycans, after the exoglycosidases have trimmed them to their core. However, for removal of intact O-glycan chains a researcher must still use chemical methods.

    References

    1. Kakehi K., et al (1994) J Chromatogr A 680, 209–215. PMID: 7952002
    2. Royle L., et al (2002) Anal Biochem 304, 70–90. PMID: 11969191
    3. Maley, F. et al. (1989) Anal. Biochem., 180, 195-204. PMID: 2510544
    4. Koutsioulis, D. et al (2008) Glycobiology , 18, 799-805. PMID: 18635885
    • Identification and Characterization of Protein Glycosylation

      Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries both N-glycans and O-glycans in this video. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.

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    • Overview of Glycobiology

      Learn about the core sequences and common modifications of N-linked and O-linked glycans in this video. Analysis of these glycans and/or peptide portions of the glycoprotein can be accomplished with the use of deglycosylation enzymes, which are explained in detail. Unlike other chemical deglycosylation methods, enzymatic treatment is much gentler and can provide complete sugar removal with no protein degradation.

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    • Elucidating the Complexity of Heparin Oligosaccharide Analysis

      Learn more about the structural elucidation of complex and diverse heparin oligosaccharides using Bacteroides Heparinase I, II and III in combination with downstream mass spec analysis.

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    FAQs for Removal of N-Linked & O-Linked Glycans from Glycoproteins