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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
Native proteins with core 1 O-glycosylation
2 h to O/N (16-18 h) reaction
Removal of sialic acids using SialEXO™ (included) is required for optimal performance
Digests N-terminally of core 1 O-glycosylated Ser and Thr residues
Product Formats
OpeRATOR™ Lyophilized
Lyophilized enzyme for digestion of mucin-type O-glycoproteins and peptides
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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.
Applications
Combining multiple digestions for O-glycan site mapping with glycan structure analysis for complete characterization of heavily O-glycosylated proteins.
Broad O-glycan substrate acceptance efficiently generating informative O-glycopeptides while retaining glycan structure information.
Precise O-glycan site mapping by generating small, MS-friendly O-glycopeptides and improved confidence through overlapping sequence coverage.
Simple and direct O-glycan site identification by site-specific digestion, generating fragments which can be used for high-resolution middle-level analysis.
Citations
Resources
How OpeRATOR™ Works
Watch video
AppNote OpeRATOR™ for Determination of O-glycan Sites
Download Application Notes
AppNote OpeRATOR™ for Automated O-glycopeptide Profile Monitoring
Download Application Notes
SmartEnzymes™ for the Glycosylation Charcterization of Glycoproteins
Download Scientific Poster
SmartEnzymes™ for O-glycosylation Analysis using Mass Spectrometry
Download Scientific Poster
SmartEnzymes™ for O-glycosylation Analysis using LC-MS
Download Scientific Poster
FAQ and Support
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.
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.
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.
Will OpeRATOR work on simple hexose or pentose?
No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.
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.
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Genovis products are intended for research use only. They are not intended to be used for therapeutic or diagnostic purposes in humans or animals. All goods and services are sold subject to Genovis’ General Terms and Conditions of Sale.
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