glyco application

Glycoprotein Analysis

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. A researcher must still use chemical methods.

Chemical methods for deglycosylation:

  • β-elimination with mild alkali (1) or mild hydrazinolysis (2)
  • These methods often result in the degradation of the protein

Enzymatic methods for deglycosylation for N-glycans:

  • Complete removal of the sugar with no protein degradation (3)
  • PNGase F has the broadest specificity
  • Other endoglycosidases are more specific with regards to the type of N-glycan they cleave
  • A simple method to determine if a glycoprotein has high mannose, complex or hybrid N-glycans is to run three reactions:

– An untreated control with just the glycoprotein and no enzyme,

– The glycoprotein treated with PNGase F, Recombinant, and

– The glycoprotein treated with Endo Hf.

Following incubation, the three samples are analyzed using SDS-PAGE and western blot, if needed.  If a shift is observed with the Endo Hf treated sample relative to the untreated control, then the glycoprotein has high mannose N-glycans.  If a shift is seen with the PNGase F sample but not with the Endo Hf sample then the glycoprotein has either complex or hybrid N-glycans, but no high mannose N-glycans.

Enzymatic methods for removal of O-glycans

  • There are no general endoglycosidases for the complete removal of O-glycans.  However, the broadest specificity O-glycosidase cleaves core 1 and core 3 O-glycans (4)
  • Using O-glycosidase with an appropriate mix of exoglycosidase allows the removal of large O-glycans.  
 

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

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FAQs for Glycoprotein Analysis
Protocols for Glycoprotein Analysis
Application Notes Glycoprotein Analysis
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