Highly Efficient Hydrolysis of Core 1 O-glycans using OglyZOR™

Performance Activity

Efficient removal of core 1 O-glycans from complex glycoproteins to reduce heterogeneity and improve mass spectrometry characterization.

O-glycans are important post-translational modifications that contribute to protein structure, stability, and biological function, while also introducing significant molecular heterogeneity. Their selective removal can simplify analytical profiles, improving resolution and accuracy in protein characterization techniques such as mass spectrometry. Etanercept is an Fc-fusion protein made up of an Fc-domain from a human IgG1 and the TNFα receptor (TNFR). The hinge region of the TNFR domain is decorated with up to 13 mucin-type O-glycans on each half of the protein and this makes etanercept a highly challenging protein to analyse at the intact or subunit level.

Here, we demonstrate efficient removal of core 1 O-glycans using OglyZOR in comparison to an O-glycosidase from another source. Both intact etanercept, and the isolated TNFR domain were incubated with OglyZOR and SialEXO, as well as an alternative O-glycosidase, for 4 hours and analysed by SDS-PAGE. Etanercept contains primarily sialylated core 1 O-glycan structures and on both the intact etanercept and the isolated TNFR domain we can see the mass shift associated with the O-glycans being removed. Co-incubating with SialEXO to remove the sialic acid meant that the OglyZOR enzyme efficiently hydrolyzed the O-glycans from both samples.

Improved removal of core 1 O-glycans using OglyZOR

Hydrolysis of core 1 O-glycan structures on native glycoproteins in a highly efficient manner significantly reduces overall molecular heterogeneity and simplifies complex glycoform profiles. This makes OglyZOR the ideal tool for streamlined sample preparation prior to mass spectrometry, improving spectral clarity and enabling more confident identification and characterisation of underlying protein modifications, and variant forms.

O-glycan removal reduces protein heterogeneity for more confident analysis of protein modifications

Improved O-glycan removal using OglyZOR. SDS-PAGE analysis of etanercept TNFR domain (left) and native intact etanercept (right) which were incubated with OglyZOR and SialEXO and an alternative O-glycosidase for 4 hours. The decrease in observed mass is due to the removal of O-glycans. OglyZOR removes the O-glycans much more efficiently than the alternative O-glycosidase, which can be seen most clearly in the TNFR sample.

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