OpeRATOR site-specifically digests proteins and peptides carrying mucin-type O-glycans



OpeRATOR (OgpA) is an O-glycan-specific protease that digests the protein backbone of proteins and peptides carrying core 1 mucin-type O-glycans. The OpeRATOR enzyme digests N-terminally of the O-glycosylated serine (Ser) or threonine (Thr) residue for O-glycan profiling and mapping of O-glycan sites. OpeRATOR has limited activity on core 3 O-glycosylated sites and the performance is optimal after desialylation using SialEXO. OpeRATOR is available as lyophilized powder for digestion of 2 mg of O-glycoprotein and supplied with lyophilized SialEXO for preparative desialylation.

Detailed Information


Product Group


, ,

Product Composition

Digestion Site



Reaction Conditions

Digestion Buffers

Buffer Compatibility



Unit Definition


OpeRATOR is a metalloprotease and as such highly sensitive to chelating agents such as EDTA. Concentrations >5mM lead to complete inhibition of the enzyme. OpeRATOR activity is also moderately inhibited by ZnCl2.

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

Yes, the enzymes work in combination and can be used to desialylate and digest an O-glycosylated protein.

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.

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.

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.

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

You may also be interested in…