NEW SmartEnzyme – Immobilized PNGase F for Fast N-glycan Hydrolysis!
Genovis launches Immobilized PNGase F, a resin with the PNGase F enzyme covalently coupled to agarose beads for hydrolysis of N-glycans on antibodies, fusion proteins and other N-glycosylated proteins in a convenient spin column format.
Benefits of using Immobilized PNGase F
- Increased enzyme to substrate ratio for faster deglycosylation
- Reduce enzyme peaks in the final sample
- Easy-to-use microspin columns with capacity of 200 ug glycoprotein
- Robust and handles both native and denaturing conditions
The Fc N-glycan play a crucial role in the effector function of antibodies but may add complexity to protein characterization assays. Removal of N-glycans from glycoproteins is widely used for sample preparation for MS analysis – to reduce the protein heterogeneity and enable released glycan analysis – and to study the functional role of the N-glycans.
The activity of PNGase F on different glycoproteins varies depending on the character of the glycoprotein, and longer incubation times or denaturing conditions may be required to reach complete removal of N-glycans. To demonstrate the deglycosylation ability of Immobilized PNGase F on various substrates, a selection of glycoproteins was processed with Immobilized PNGase F. The 6 N-glycans of the Fc-fusion protein aflibercept was successfully deglycosylated by Immobilized PNGase F in 30 min under denaturing reaction conditions. Complete deglycosylation was also achieved under native condition but required overnight (18 h) incubation with Immobilized PNGase F. Using both denaturing and reducing reaction condition, the commonly used glycoprotein standard RNase B was completely deglycosylated in 15 min. The Fc-glycans on the therapeutic antibody cetuximab were efficiently removed by Immobilized PNGase F within one hour using native reaction conditions, however, denaturing conditions were required to remove the N-glycans in the Fab region of the antibody.
Key characteristics and deconvoluted mass spectra or SDS-PAGE assay of a selection of glycoproteins deglycosylated using Immobilized PNGase F. The table above specifies important characteristics of the examined glycoproteins, the Immobilized PNGase F reaction conditions used for the deglycosylation and the sample preparations performed before analysis. Abatacept was analyzed by reverse-phase LC-MS on a Waters BioAccord system equipped with a Waters BioResolve RP mAb column (2.1 x 50 mm). RNase B and cetuximab were analyzed on SDS-PAGE. The mass shifts in the deconvoluted mass spectra or on the gels show the successful removal of N-glycans from the various glycoprotein substrates.
Learn more about Immobilized PNGase F for deglycosylation of glycoproteins.
Immobilized PNGase F – Lyophilized enzyme for removal of α1-2, α1-3 and α1-4 linked fucose from 2 mg glycoprotein.
Immobilized PNGase F Denaturing – Immobilized enzyme for hydrolysis of N-glycans from glycoproteins in spin columns under denaturing conditions (RapiGest™* SF Surfactant from Waters™ included)
* RapiGest™ SF Surfactant from Waters Corporation is included in Immobilized PNGase F Denaturing. RapiGest™ is a trademark of Waters Corporation.