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), PubMedID: 27033327, DOI:
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at firstname.lastname@example.org.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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.