OpeRATOR™ – O-glycan-specific Protein Digestion

OpeRATOR is an O-glycan-specific protease that digests proteins carrying core 1 O-glycans, N-terminally of the glycosylated (core 1) Ser and Thr residues.

SmartEnzymes™

With OpeRATOR, glycopeptides carrying O-glycans are generated, and the enzyme enables O-glycan profiling, O-glycopeptide mapping and site occupancy determination, as well as middle-level approaches using MS analysis.

Why OpeRATOR™?

  • Reliable O-glycan-specific protease – digests N-terminally of Ser/Thr glycosylation sites
  • Robust digestion for enhanced characterization of complex biopharmaceuticals
  • High specificity – confident glycan site determination and mapping

  • Target

    Native proteins with core 1 O-glycosylation

  • Time

    2 h to O/N (16-18 h) reaction

  • Box

    Removal of sialic acids using SialEXO™ (included) is required for optimal performance

  • Digestion Site

    Digests N-terminally of core 1 O-glycosylated Ser and Thr residues

Product Formats

About OpeRATOR™

OpeRATOR is an endoprotease that catalyzes the hydrolysis of the peptide bonds of native mucin-type O-glycosylated proteins that are 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 residues.

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 most active towards sites with asialylated core1 O-glycans. It also digests sialylated core 1 and core 3, but to a much lower extent. Removal of sialic acids is required for optimal performance, and therefore, SialEXO Lyophilized (sialidase mix) is included in the purchase.

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 SialEXO Lyophilized also contain His-tags, and the molecular weights of these components are 43 kDa and 66 kDa, respectively.

OpeRATOR is an O-glycan-specific protease that digests proteins carrying core 1 O-glycans, N-terminally of the glycosylated (core 1) Ser and Thr residues.

Applications

Citations

Resources

FAQ and Support

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

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.

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

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.

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

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