NEW! We’re Launching ImpaRATOR™!

We are excited to launch ImpaRATOR, an O-glycan-dependent protease that digests proteins carrying mucin-type O-glycans, including sialylated species, N-terminally of glycosylated Ser and Thr residues!
 
The enzyme 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.
 

• 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

ImpaRATOR™ Accepts a Wide Range of O-glycan Species

To demonstrate the broad glycan specificity of ImpaRATOR, three samples of etanercept with different glycan compositions were prepared: (i) the untreated glycoprotein carrying a mixture of mono- and di-sialylated core 1 structures, (ii) the glycoprotein treated with SialEXO to remove sialic acids and generate naked core 1 structures, and (iii) the glycoprotein treated with SialEXO and GalactEXO to generate single GalNAc residues, also known as Tn antigen. The three samples were treated with ImpaRATOR and FabRICATOR in a one-pot reaction. As a comparison, SialEXO-treated etanercept was treated with OpeRATOR and FabRICATOR. The resulting peptides were reduced, alkylated and analyzed by HILIC-MS. FabRICATOR was included in the reaction to separate the Fc region from the most C-terminal glycopeptide to facilitate its characterization.
 
Analysis of the ImpaRATOR-treated samples showed that ImpaRATOR digested all three substrates well and generated similar glycopeptides, but with the expected different glycan species. The sample treated only with ImpaRATOR contained more variants than the other samples since each peptide was detected with several glycan structures. The observed glycan species were mainly mono- and di-sialylated core 1 structures, which is in accordance with the expected ones for etanercept. The SialEXO-pretreated samples digested with ImpaRATOR or OpeRATOR both resulted in peptides with naked core 1 structures, with the exception of peptide T205-P207 carrying the Tn antigen observed in the ImpaRATOR-digested sample. Etanercept pretreated with SialEXO and GalactEXO followed by digestion with ImpaRATOR resulted in peptides with only the Tn antigen. Together, this data demonstrates the high glycan substrate acceptance of ImpaRATOR.
 

 
ImpaRATOR activity on etanercept with different glycan compositions. Etanercept a) untreated, b) treated with SialEXO, or c) treated with SialEXO and GalactEXO was digested by ImpaRATOR and FabRICATOR. d) Etanercept pretreated with SialEXO was digested with OpeRATOR and FabRICATOR. Peptides were reduced, alkylated and separated by HILIC (ACQUITY UPLC Glycoprotein Amide column, 300 Å, 1.7 μm, 2.1 mm × 150 mm) and analyzed on a Waters™ BioAccord™ LC-MS system. Shaded peaks indicate O-glycopeptides shared in (b) and (d).

ImpaRATOR Lyophilized – Process 2 mg O-glycoprotein.