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.
FAQs for Removal of N-Linked & O-Linked Glycans from Glycoproteins
- What happens to my protein after deglycosylation?
- Does enzymatic N-and O-glycan removal preserve protein structural integrity and function?
- Do the Remove-iT glycosidases have the same specificity as enzymes without a tag?
- Can endoglycosidases be removed from the reaction?
- What is a good endoglycosidase substrate?
- Can glycosidases be used in combination for extensive digestion?
- Do I need to deglycosylate my protein sample for proteomic analysis?
- What are glycosidases and their uses?
Protocols for Removal of N-Linked & O-Linked Glycans from Glycoproteins
- α1-2,4,6 Fucosidase O Digestion of Released Labeled Glycans Protocol
- O-Glycosidase (P0733)
- O-Glycosidase Application Note 1 (P0733)
- Endo D Removal Magnetic Chitin Bead Protocol (P0742)
- Endo F2 Reaction Protocol (P0772)
- Endo H/Endo Hf Protocol
- Endo S Removal Magnetic Chitin Bead Protocol (P0741)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- Glycan SPE C18 and Graphitized Carbon Protocols (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0711)
- Intact Protein LS-ESI-TOF Protocol (P0710)
- PNGase F Protocol
- Protocol for α1-3,6 Galactosidase (P0731)
- Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol
- Rapid PNGase F (non-reducing format) (P0711) SDS-PAGE Protocol
- Rapid PNGase F Antibody Standard Protocol (P6043)
- Rapid PNGase F by SDS-PAGE Protocol (P0710)
- Rapid PNGase F Protocols (P0710)
- Reaction Conditions for Endo D (P0742)
- Reaction Conditions for Endo S (P0741)
- Reaction Conditions for PNGase A (P0707)
- Reaction Conditions for Remove-iT® PNGase F (P0706)
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Removal of Endo F2 by Magnetic Beads (P0772)
- Removal of terminal N-acetylglucosamine from the biantennary N-linked sugars of IgG
- Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
- RNase B Deglycosylation Protocol (P7817)
- Typical Reaction Conditions α-N-Acetylgalactosaminidase (P0734)
- Typical Reaction Conditions (P0732)
- Typical Reaction Conditions for α1-2 Fucosidase (P0724)
- Typical Reaction Conditions for α1-2, 3, 4, 6 Fucosidase (P0748)
- Typical Reaction Conditions for α1-2,3,6 Mannosidase (P0768)
- Typical Reaction Conditions for α1-3, 4 Fucosidase (P0769)
- Typical Reaction Conditions for α1-3, 4, 6 Galactosidase (P0747)
- Typical Reaction Conditions for α1-3,6 Galactosidase (P0731)
- Typical Reaction Conditions for α2-3 Neuraminidase S (P0743)
- Typical Reaction Conditions for α2-3,6,8 Neuraminidase (P0720)
- Typical Reaction Conditions for α2-3,6,8,9 Neuraminidase A (P0722)
- Typical Reaction Conditions for β-N-Acetylglucosaminidase S (P0744)
- Typical Reaction Conditions for β1-3 Galactosidase (P0726)
- Typical Reaction Conditions for β1-3,4 Galactosidase Reaction Protocol (P0746)
- Typical Reaction Conditions for β1-4 Galactosidase S (P0745)
- Typical Reaction Conditions for β1-4 Galactosidase (P0730)
- Typical Reaction Conditions for β-N-Acetylhexosaminidasef (P0721)
- Typical Reaction Conditions for α1-2,3 Mannosidase (P0729)
- Typical Reaction Conditions for Endo F3 Protocol (P0771)
- O-Glycosidase Application Note 1
Other Tools & Resources
Applications of Deglycosylation
There are a number of reasons to deglycosylate a glycoprotein, including:
- To remove heterogeneity in glycoproteins for X-ray crystallographic analysis
- To remove carbohydrate epitopes from antigens
- To enhance or reduce blood clearance rates of glycoprotein therapeutics
- To investigate the role of carbohydrates in enzyme activity and solubility
- To investigate ligand binding
- For quality control of glycoprotein pharmaceuticals
- To simplify analysis of the peptide portion of the glycoprotein
- To simplify the analysis of the glycan component via glycan sequencing
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