Removal of N-glycans from Glycoproteins

Efficient N-glycan Removal In a Convenient Spin Column Format

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. PNGase F (Peptide N-glycosidase F) is a glycoamidase hydrolyzing the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. During the reaction, the asparagine residue from which the glycan is removed is deaminated to aspartic acid, and the released oligosaccharide is left intact and can be used for further analysis.
 
Immobilized PNGase F has the PNGase F enzyme covalently coupled to agarose beads in easy-to-use spin column format for efficient removal of N-glycans.



Schematic overview of the Immobilized PNGase F workflow.

PNGase F Workflow

Removal of N-glycans under Native Conditions

The Fc N-glycan plays a crucial role in the effector function of antibodies but may add complexity to protein characterization assays. Removing the Fc N-glycan with PNGase F under native conditions enables characterization of both the free N-glycan and the function and structure of the deglycosylated antibody.
To demonstrate the efficient removal of Fc N-glycans by Immobilized PNGase F under native reaction conditions, the therapeutic antibody trastuzumab was processed using the native Immobilized PNGase F workflow. The mass shift in Fig. 1 demonstrates successful removal of the Fc N-glycans on trastuzumab after incubation with Immobilized PNGase F for 1 h at 37°C, with no enzyme interfering in the analysis as compared to the sample processed with PNGase F in solution.

 

Benefits of using Immobilized PNGase F

  • Increase 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

 

 

 

 

 

Figure 1. TIC chromatogram (top) and deconvoluted mass spectra (bottom) of the Fc/2 fragment of trastuzumab treated with Immobilized PNGase F under native conditions or PNGase F in solution. Trastuzumab was incubated for 1 h with Immobilized PNGase using native reaction conditions or PNGase F in-solution for 1 h at 37°. The deglycosylated antibody was digested with FabRICATOR, reduced and analyzed by reverse-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve™ RP mAb column (2.1 x 50 mm). The TIC chromatogram clearly shows the absence of the PNGase F enzyme in the sample treated with Immobilized PNGase F.

Denaturing Conditions Speed up the Process

Some glycosylation sites are poorly accessible for PNGase F and the deglycosylation reaction is slow or inhibited completely by steric hindrance. Denaturation of the glycoprotein increases the rate of deglycosylation. To demonstrate the increase in reaction speed under denaturing conditions, the fusion protein abatacept was processed with Immobilized PNGase F using either the native or the RapiGest denaturing workflow (Fig. 2). Native conditions allowed for removal of N-glycans without any additives or increased temperature but required overnight (18 h) incubation for complete deglycosylation. Removal of the N-glycans using denaturing conditions was complete within 30 minutes. As can be seen from the MS spectra, all N-glycans are removed while the O-glycans are left intact. The intact protein is complex to analyze, but after Immobilized PNGase F treatment, the complexity of the sample was greatly reduced.

Figure 2. Deconvoluted mass spectra of abatacept (top panel) treated with Immobilized PNGase F under denaturing (middle panel) or native conditions (bottom panel). The protein was incubated for 18 h with Immobilized PNGase F using native reaction conditions or 30 min using denaturing conditions. The deglycosylated protein samples were reduced and analyzed by reverse-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve™ RP mAb column (2.1 x 50 mm). The mass shifts show the complete removal of N-glycans, leaving only the O-glycoforms.
 
 

Rapid N-glycan Removal using Immobilized PNGase F Denaturing

To demonstrate the deglycosylation ability of Immobilized PNGase F on various substrates, a selection of glycoproteins was processed with ImmobilizedPNGase F (Fig. 3). All N-glycans on trastuzumab and etanercept, and the Fc-glycans on cetuximab were efficiently removed by Immobilized PNGase F within one hour using native reaction conditions. When adding RapiGest™ and performing the reaction under denaturing conditions, all N-glycans – including the Fab-glycans – of cetuximab were completely removed within 15 min. The more complex Fc-fusion proteins abatacept and aflibercept were successfully deglycosylated by Immobilized PNGase F in 30 min under denaturing reaction conditions. Using both denaturing and reducing reaction condition, the commonly used glycoprotein standard RNase B was completely deglycosylated in 15 min. The Immobilized PNGase F-processed samples generated under both native or denatured conditions are compatible with LC-MS analysis.

Figure 3. Key characteristics and 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 SDS-PAGE analysis. The mass shifts on the gels show the successful removal of N-glycans from the various glycoprotein substrates.
 
 


 
* RapiGest™ SF Surfactant from Waters Corporation is included in Immobilized PNGase F Denaturing. RapiGest™ is a trademark of Waters Corporation.

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