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Detailed Information

OpeRATOR is an endoprotease that catalyzes the hydrolysis of peptide bonds in native mucin-type O-glycosylated proteins in direct proximity of the O-glycans. With high specificity, the endoprotease digests proteins at the N-terminus of O-glycans at serine or threonine. O-glycans of mucin-type are required for OpeRATOR activity and the enzyme will not digest glycoproteins with only N-linked glycans. The enzyme is active on O-glycan proteins with or without sialic acids, although the activity is higher on O-glycan proteins where sialic acids have been removed. OpeRATOR can be used together with SialEXO (sialidase), which is included in the box.

OpeRATOR is derived from Akkermansia muciniphila and expressed in E. coli. The enzyme contains a His-tag and the molecular weight is 42 kDa. SialEXO is derived from Akkermansia muciniphila and expressed in E. coli. The enzymes in the SialEXO also contain a His-tag and the molecular weights of these components are 43 kDa and 66 kDa, respectively.

Unit Definition

One unit digests ≥ 90% of 1 µg glycoprotein (TNFαR) when incubated together with one unit of SialEXO in 20 mM Tris pH 6.8 at 37 °C for 2 h.

Content and Storage

The OpeRATOR box includes:

1 vial of OpeRATOR enzyme (G1-OP1-020) supplied lyophilized in TBS pH 7.6, with no preservatives added.
1 vial of SialEXO (G1-SM1-020) supplied lyophilized in TBS pH 7.6, with no preservatives added.

The box is shipped at ambient temperature, and should be stored at -20°C upon arrival.. After reconstitution the enzymes are stable for 1 month at +4-8 °C.

FAQ and Support

Popular FAQ

Can OpeRATOR digest consecutive O-glycosylated sites fully?

The relative abundance and site occupancy of O-glycosylation may vary a lot in a glycoprotein sample, and this will result in the generation of peptides with variable sizes. In some cases, the peptides contain more than one O-glycan, i.e. OpeRATOR does not digest at every site in every molecule. However, the missed digestion sites differ from molecule to molecule, and the compiled data will provide valuable information on the O-glycosylated sites.

Does the enzyme require the sample to be in the native fold or would denaturation help?

If digestion is insufficient, it could be caused by the O-glycans having a sterically inaccessible location on the sample that the enzyme cannot reach. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, rebuffering, and then digestion with SialEXO and OpeRATOR.

Is removal of sialic acids really needed?

The activity of OpeRATOR is significantly decreased in the presence of sialic acids. We recommend using SialEXO to remove the sialic acids. If this is not an option, then we suggest trying to digest both with and without SialEXO to evaluate the impact for your specific samples and requirements.

Will OpeRATOR work on simple hexose or pentose?

No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.

Can OglyZOR be used to deglycosylate peptides generated with OpeRATOR?

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

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