Fast PNGase F, Completing Deglycosylation in Just 10 Minutes

With the rapid development of glycoproteomics and antibody drug research, glycosylation modification has also attracted much attention. The contact between cells and cells, and between cells and pathogens is mainly completed through the interaction of sugars and proteins, and glycosylation determines the adhesion properties of glycoconjugates. In IgG, the conserved N-linked glycosylation at Asn297 in the Fc region is also crucial for its activity. In addition, some antibodies also have additional N-glycans, which, together with the conserved site at Asn297 in the Fc region, affect the recognition, half-life, and immune response of the antibody.

Protein glycosylation is a complex post-translational modification that involves the connection of glycan chains at specific sites on proteins. Depending on the type of glycan chain, glycoproteins are divided into N-linked glycoproteins, O-linked glycoproteins, etc.; moreover, protein glycosylation is also affected by the host cell type and fermentation conditions (such as culture medium, pH value, temperature, etc.). Therefore, glycoproteins usually have heterogeneity in glycosylation patterns, including glycosylation sites, glycosylation levels, and the specific structure of glycan chains, making glycan chain analysis and structural characterization work extremely challenging. To obtain the maximum amount of glycan chain structural information, the main strategy of glycan chain analysis is to first release the glycan chains from the proteins and then carry out detailed analysis and characterization. In this strategy, an efficient, accurate, and stable deglycosylation method is crucial.

Enzymatic methods are currently widely used for deglycosylation.

Glycoproteins can be classified into N-linked and O-linked glycopeptides based on their types of glycan chains. For N-linked glycopeptides, commonly used enzymes include peptide N-glycosidase F (PNGase F), endoglycosidase H (Endo H) and endoglycosidase S (Endo S). PNGase F hydrolyzes the GlcNAc-Asn bond and can remove high mannose, complex, and hybrid glycan chains. Endo H cleaves glycosidic bonds within the N-glycan chain pentasaccharide core. Endo S specifically cleaves N-linked glycans from the chitobiose core of the heavy chain of native IgG.

Figure 1. Cutting site of PNGase F, Endo H and Endo S.

Among them, PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides in glycoproteins, which can cleave high-mannose, hybrid, and complex oligosaccharide glycoproteins connected by asparagine, specifically removing N-linked glycans. The cleavage site is: the amide bond between the innermost N-acetylglucosamine (GlcNAc) and the asparagine residue, while converting the asparagine on the protein after enzymatic hydrolysis to aspartic acid.

When α1-6 fucose is located at the GlcNAc core, PNGase F can also cut; only when α1-3 fucose is located at the GlcNAc core (common in plant and insect glycoproteins), PNGase F cannot cut.

However, the conventional PNGase F enzymatic reaction requires several hours to release antibody N-glycans, and due to the preference for glycan release, incomplete deglycosylation may lead to biased results, and the obtained glycan distribution may not represent the correct composition of therapeutic antibodies. Therefore, obtaining an accurate N-glycan profile as quickly as possible is crucial for glycosylation monitoring during the production process of antibodies and antibody fusion proteins and other biotherapeutic agents.

Fast PNGase F

Fast PNGase F is an optimized recombinant reagent that can rapidly and completely deglycosylate antibodies, immunoglobulins, fusion proteins, and other glycoproteins in a few minutes. This enzyme can quickly and without preference remove all N-glycans and can be directly used for downstream chromatography or mass spectrometry analysis. Yeasen Fast PNGase F, N-glycosidase F (Fast Version) simplifies the experimental process, reducing experimental time while ensuring sensitivity and repeatability.

Product Features:

Fast: Complete and rapid deglycosylation in a few minutes.

High Purity: No protease, glycosidase contamination, purity ≥95%.

Non-selective: Quickly and without preference remove all N-glycans.

Good Compatibility: Directly used for downstream chromatography or mass spectrometry analysis.

Test Data:

One-step protein deglycosylation:

10 ug substrate/100 ug antibody and ddH2O mixed to a total volume of 16 uL;
Add 4 μL of Fast PNGase F Buffer (5x), final volume 20 uL;
Add 1 μL of Fast PNGase F.
Incubate at 50°C for 10 minutes.

Figure 2. One-step enzymatic cleavage effect on protein substrate (A) and antibody (B).

1: Protein MarkerCat#20350ES) 2: Competitor + substrate or antibody 3: Competitor + substrate or antibody 4: Yeasen Fast PNGase F 5: Yeasen Fast PNGase F6: Substrate or antibody 7: Yeasen Fast PNGase F 8: Competitor

Two-step protein deglycosylation:

10 ug substrate/100 ug antibody and ddH2O mixed to a total volume of 16 uL;
Add 4 μL of Fast PNGase F Buffer (5x), final volume 20 uL;
Incubate at 80°C for 2 minutes, cool to room temperature.
Add 1 μL of Fast PNGase F.
Incubate at 50°C for 10 minutes.

Figure 3. Two-step enzymatic cleavage effect on protein substrate (A) and antibody (B).

Conclusion:

Yeasen Fast PNGase F has the same or higher enzymatic cleavage efficiency as imported competitors under the same reaction conditions;
It is recommended to perform a two-step enzymatic cleavage to ensure higher enzymatic cleavage efficiency. Some antibodies (such as Fab N-glycosylation) require a preheating step

for efficient deglycosylation.

In addition, Yeasen also provides regular PNGase F(Cat#20407ES, specific activity: 100000 U/mL), and other types of glycosidases, such as Endo H glycosidase H (Cat#20414ES), Endo S glycosidase S (Cat#20413ES).

Purchase Guide

Product umber	20406ES	20407ES	20414ES	20413ES
Product Name	Fast PNGase F	PNGase F	Endo H	Endo S
Source	Yeast recombinant expression	Yeast recombinant expression	Yeast recombinant expression	E.coli recombinant expression
Specific Activity	Not applicable	100000U/mL	1000000U/mL	8000 U/mg
Cleavage Site	Almost all N-linked glycans	Almost all N-linked glycans	N-linked high mannose glycans	Cleaves within the core disaccharide structure of IgG heavy chain
Digestion Time	10 min	1-3 h	1-3 h	1 h
Glycosylation	Suitable	Suitable	Suitable	Suitable
Protein Structure	Suitable	Suitable	Suitable	Suitable

ordering Information

Product Name	Product Number	Specification
Fast PNGase F (Glycerol-free)	20406ES20/50	20 T/50 T
PNGase F	20407ES01/02	15000 U /75000 U
Endo H	20414ES92/97	10000 U /50000 U
Endo S	20413ES80/90	1000 U/5 x 1000 U