ImpaRATOR™ – O-glycan-specific Protein Digestion

ImpaRATOR is an O-glycan-dependent protease that digests proteins carrying mucin-type O-glycans, including sialylated species, N-terminally of glycosylated Ser and Thr residues.

SmartEnzymes™

ImpaRATOR generates glycopeptides carrying O-glycans, which enables O-glycan profiling, site occupancy determination and O-glycopeptide mapping as well as middle-level approaches using LC-MS analysis.

Why ImpaRATOR™?

  • Broad specificity – accepts a wide range of O-glycan variants, including sialylated species
  • Reliable O-glycan-specific protease – digests N-terminally of Ser/Thr glycosylation sites
  • Robust digestion for enhanced characterization of complex biopharmaceuticals

  • Target

    Native proteins with mucin-type O-glycosylation, including sialylated species

  • Time

    2 h reaction (complex substrates may require longer incubation times)

  • Box

    No need for reducing agents or co-factors

  • Digestion Site

    N-terminally of O-glycosylated Ser and Thr

Product Formats

About ImpaRATOR™

ImpaRATOR is an O-glycan-dependent protease that catalyzes the hydrolysis of the peptide bond adjacent to glycosylated serine or threonine residues in glycoproteins and glycopeptides. It cleaves N-terminally of serine or threonine residues modified with mucin-type O-glycans, including sialylated species.

Mucin-type O-glycans are required for ImpaRATOR activity and the enzyme will not digest unmodified serine or threonine residues, or at N-glycosylation sites of glycoproteins. The enzyme accepts a broad range of O-glycan structures, including sialylated core 1 and core 2 structures as well as the Tn antigen.

ImpaRATOR is derived from Pseudomonas aeruginosa and expressed in E. coli. The enzyme contains a His-tag and has a molecular weight of 97 kDa.

Applications

Resources

FAQ and Support

ImpaRATOR has a broad activity towards different O-glycan structures; however, the enzyme has limited activity towards sites with two adjacent O-glycosylated Ser/Thr residues. For complete information about O-glycan sites in glycoprotein substrates containing several sites with two adjacent O-glycosylated Ser/Thr residues, OpeRATOR may be a better option.

ImpaRATOR is a metalloprotease and thereby sensitive to chelating agents such as EDTA. Concentrations > 1 mM EDTA results in complete inhibition of the enzyme. In addition, the ImpaRATOR activity is inhibited by reducing agents and detergents.

Insufficient digestion during non-denaturing conditions can be caused by O-glycosylation sites within the glycoprotein inaccessible to the enzyme. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, buffer-exchange, and then digestion with ImpaRATOR. If detergent is added, make sure to include a detergent removal step prior ImpaRATOR digestion.

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