Can OglyZOR be used to deglycosylate peptides generated with OpeRATOR?
Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.
Are there any known inhibitors of OpeRATOR?
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.
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 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.
Can OpeRATOR and SialEXO be used in the same reaction?
Yes, the enzymes work in combination and can be used to desialylate and digest an O-glycosylated protein.