Efficient O-glycan Hydrolysis

Hydrolysis of Core 1 type O-glycans

O-glycosylation is not only difficult to study, its natural heterogeneity can also cause problems in other analyses. For example, mass spectrometric analysis of heavily glycosylated proteins at the intact or middle-level can be very difficult due to the many different glycoforms present in the sample. Solutions for enzymatic removal of N-glycans have been available for many years and PNGase F is a very well-established tool. Removal of O-glycans can be more challenging and existing chemical methods for O-glycan release often cause major modifications to the protein and are therefore less suited for down- stream analysis.


OglyZOR Requires Removal of Sialic Acids

The OglyZOR enzyme efficiently hydrolyzes core 1 disaccharides (Gal-β1,3-GalNAc) from native glycoproteins. This makes the enzymes suitable for preparation of samples prior to LC-MS analysis for identification of other PTMs or confirmation of amino acid sequences. To investigate the OglyZOR activity on native glycoproteins, the enzyme was incubated with etanercept, TNFR and abatacept for 1 h at 37°C with or without SialEXO. When the sialic acids are removed, the OglyZOR enzyme efficiently hydrolyzes the O-glycans from all three substrates (Fig. 1).


Poster PEGS 2019 - Deglycosylation of Biopharmaceuticals

Figure 1. Efficient O-glycan removal from native glycoproteins using OglyZOR and SialEXO.


Total Deglycosylation of O-glycosylated Biopharmaceuticals

O-glycosylated biopharmaceuticals such as etanercept are difficult to characterize. To enable determination of intact mass by mass spectrometry , we performed enzymatic total deglycosylation.  N-glycans were cleaved by PNGase F, while the O-glycans could be removed using OglyZOR together with SialEXO. The resulting deglycosylated protein could then be digested with FabRICATOR and analyzed by middle-level mass spectrometry. We applied this workflow for analysis of etanercept and while the untreated TNFR domain was too complex to analyze (Fig. 2a), the O-glycan composition could be resolved and compared after treatment with PNGase F (Fig. 2c).


Figure 2.
Deconvoluted TIC spectra of TNFR and Fc subunits of etanercept from three commercially available preparations. a) The TNFR subunit carrying both N- and O-glycans is complex. 
b) After removal of all O-glycans (SialEXO, OglyZOR) the combined major N-glycans on the two sites, G2F G2 and G2F G2F (sialidase treated) was verified. c) Complete glycan removal (OglyZOR, SialEXO, PNGaseF) provided information of the primary structure of the TNRF part. N-terminal loss, –L (2-256) is visualized. A fraction of the deglycosylated subunits revealed masses +HexNAc indicating presence of incomplete core 1, not removed by OglyZOR.