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.
- What are glycosidases and their uses?
- What is a good endoglycosidase substrate?
- Can endoglycosidases be removed from the reaction?
- Can glycosidases be used in combination for extensive digestion?
- Do I need to deglycosylate my protein sample for proteomic analysis?
- Do the Remove-iT glycosidases have the same specificity as enzymes without a tag?
- What happens to my protein after deglycosylation?
- Does enzymatic N-and O-glycan removal preserve protein structural integrity and function?
- O-Glycosidase Application Note 1 (P0733)
- O-Glycosidase (P0733)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- Protocol for α1-3,6 Galactosidase (P0731)
- Removal of terminal N-acetylglucosamine from the biantennary N-linked sugars of IgG
- Typical Reaction Conditions for α2-3,6,8 Neuraminidase (P0720)
- Typical Reaction Conditions for α1-2 Fucosidase (P0724)
- Endo H/Endo Hf Protocol
- Typical Reaction Conditions (P0732)
- Typical Reaction Conditions for β-N-Acetylhexosaminidasef (P0721)
- Typical Reaction Conditions for β1-3 Galactosidase (P0726)
- Typical Reaction Conditions for α1-2,3 Mannosidase (P0729)
- Typical Reaction Conditions for β1-4 Galactosidase (P0730)
- Typical Reaction Conditions for α1-3,6 Galactosidase (P0731)
- Typical Reaction Conditions α-N-Acetylgalactosaminidase (P0734)
- Reaction Conditions for Remove-iT® PNGase F (P0706)
- Reaction Conditions for Endo S (P0741)
- Endo S Removal Magnetic Chitin Bead Protocol (P0741)
- Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
- Reaction Conditions for Endo D (P0742)
- Endo D Removal Magnetic Chitin Bead Protocol (P0742)
- RNase B Deglycosylation Protocol (P7817)
- PNGase F Protocol
- Typical Reaction Conditions for β1-4 Galactosidase S (P0745)
- Typical Reaction Conditions for α2-3,6,8,9 Neuraminidase A (P0722)
- Typical Reaction Conditions for α2-3 Neuraminidase S (P0743)
- Typical Reaction Conditions for β-N-Acetylglucosaminidase S (P0744)
- Rapid PNGase F Protocols (P0710)
- Intact Protein LS-ESI-TOF Protocol (P0710)
- Rapid PNGase F by SDS-PAGE Protocol (P0710)
- Glycan SPE C18 and Graphitized Carbon Protocols (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0710)
- Typical Reaction Conditions for α1-3, 4, 6 Galactosidase (P0747)
- Typical Reaction Conditions for α1-2, 3, 4, 6 Fucosidase (P0748)
- Typical Reaction Conditions for α1-3, 4 Fucosidase (P0769)
- Rapid PNGase F Antibody Standard Protocol (P6043)
- Glycoproteomics: Buffer Exchange Protocols (P0711)
- Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol
- Rapid PNGase F (non-reducing format) (P0711) SDS-PAGE Protocol
- Typical Reaction Conditions for β1-3,4 Galactosidase Reaction Protocol (P0746)
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Typical Reaction Conditions for Endo F3 Protocol (P0771)
- Reaction Conditions for PNGase A (P0707)
- Typical Reaction Conditions for a1-2,3,6 Mannosidase (P0768)
- Endo F2 Reaction Protocol (P0772)
- Removal of Endo F2 by Magnetic Beads (P0772)
- a1-2,4,6 Fucosidase O Digestion of Released Labeled Glycans Protocol
Using Glycosidases to Remove Trim or Modify Glycans on Therapeutic Protein
N-Glycan Composition Profiling for Quality Testing of Biotherapeutics
O-glycan analysis of therapeutic proteins enabled by O-glycoprotease
This article from European Pharmaceutical Review describes tools that can be used for O-glycan analysis of therapeutic proteins.
- Vainauskas, S., Kirk, C.H., Petralia, L., Guthrie, E.P., McLeod, E., Bielik, A., Luebbers, A., Foster, J.M., Hokke, C.H., Rudd, P.M., Shi, X., Taron, C.H (2018) A novel broad specificity fucosidase capable of core a1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes Sci Rep; PubMedID: 29934601, DOI: 1038/s41598-018-27797-0
- Albrecht, S., Vainauskas, S., Stöckmann, H., McManus, C., Taron, C.H. and Rudd, P.M. (2016) Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow. Anal Chem; May 3;88(9), 4795-802. Anal Chem.. PubMedID: 27033327
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Learn about the core sequences and common modifications of N-linked and O-linked glycans in this video. Analysis of these glycans can be accomplished with the use of deglycosylation enzymes, which can provide complete sugar removal with no protein degradation.
This one-step deglycosylation protocol is for use with New England Biolabs’ Rapid PNGase F. Learn more about Rapid PNGase F.
This two-step deglycosylation protocol is for use with New England Biolabs’ Rapid PNGase F. Learn more about Rapid PNGase F.
Behind the Paper: An engineered Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides
Minyong and Jim summarize their recent Nature Communications publication describing selective capture of N-glycans and N-glycopeptides by an engineered high affinity Fbs1 carbohydrate binding protein.