New England Biolabs, P0761S, O-Glycoprotease (IMPa)

O-Glycoprotease (IMPa) is a broad specificity protease that cleaves the peptide bonds of a glycoprotein or glycopeptide immediately N-terminal to a serine or threonine residue containing a mucin-type O-linked glycan with or without sialylation. Related Categories Protein Tools,, Proteases,, Glycobiology, Applications Glycomics and glycoproteomics,, Protein Digestion,, Glycobiology & Proteomics, Specification Unit Definition One unit of O-Glycoprotease (IMPa) will cleave > 90% of 2 pmol FAM-labelled O-glycopeptide in a total reaction volume of 20 μl in 2 hours at 37°C in 20mM Tris-HCl, pH 8.0. Storage Buffer 20 mM Tris-HCl 100 mM NaCl pH 7.5 @ 25°C Heat Inactivation 95°C for 10 minutes Molecular Weight Theoretical: 97 kDa Unit Assay Conditions Two-fold dilutions of O-Glycoprotease (IMPa) are incubated with 2μM of FAM-labelled O-glycopeptide and 20 mM Tris-HCl pH 8.0 in a 20 μl reaction. The reaction mix is incubated at 37°C for 2 hours. Separation of reaction products are determined by CE analysis. FAQ Q: Can an O-Glycoprotease (IMPa) reaction be scaled linearly to accommodate digestion of greater than 10 μg of glycoprotein? A: Yes, the O-Glycoprotease (IMPa) reaction can be scaled linearly. Typical reaction conditions recommend 1 µl of O-Glycoprotease (IMPa) per 10 µg of substrate; reactions can be scaled linearly by maintaining an enzyme:substrate ratio of 1:10. However, the exact enzyme to substrate ratio must be determined empirically as substrate purity, tight folding, degree of glycosylation and clustering of O-glycosylation (IMPa) sites may affect the digestion efficiency. Longer incubation times (18 hours) and more enzyme may be necessary in some cases. It is recommended to first test the digestion efficiency at small scale (10 µg) prior to scaling up, and to monitor the efficiency of cleavage using SDS-PAGE analysis. Q: Is O-Glycoprotease (IMPa) active in the presence of reducing agents or detergents? A: No, O-Glycoprotease (IMPa) is not active in the presence of reducing agents or detergents. Addition of 1mM DTT, or detergents such as Invitrosol (Thermo, #MS1007) and Protease Max (Promega, # V2071), completely inactivate the enzyme. If using reducing agents or detergents, the glycoprotein must be purified prior to the addition of O-Glycoprotease (IMPa). We recommend the detergent removal column HiPPR Detergent Removal Resin (Thermo catalog #88306), which should be used according to the manufacturer’s recommended instructions. Q: Is O-Glycoprotease (IMPa) active in buffers other than 20 mM Tris-HCl pH 8.0? A: Yes, the optimal pH range for O-Glycoprotease (IMPa) is pH 7.0-8.0. Enzyme activity drops significantly at pH values below 7.0 and above pH 8.0. O-Glycoprotease is 100% active in 20mM HEPES, pH 8.0. The enzyme has a 50% reduction of activity in 15-25 mM NH4CO3 and is not active in 50 mM NH4CO3. Q: Is O-Glycoprotease (IMPa) able to efficiently cleave clustered O-glycosylated sites? A: Analysis of highly clustered O-glycosylated sites can be challenging due to the diverse site occupancy (macroheterogeneity) and close proximity of O-glycosylation sites in glycoproteins. Parallel analysis of a native and denatured glycoprotein digested with O-Glycoprotease (IMPa) can help to obtain more complete information. Longer incubation times (18 hours) and more enzyme may be necessary in some cases. In addition, O-Glycoprotease (IMPa) does not cleave between two adjacent O-glycosylated amino acids. Q: I see minimal cleavage of my glycoprotein after treatment with O-Glycoprotease (IMPa). How can I optimize the reaction? A: Tightly folded glycoproteins and/or glycoproteins with clustered O-glycosylated sites can be resistant to cleavage with proteases. In these cases, denaturing the substrate prior to digestion with O-Glycoprotease (IMPa) may be necessary for optimal digestion. In addition, longer incubation times (18 hours) and more enzyme may be necessary. However, please note that detergents and additional reagents must be removed from the reaction prior to the addition of O-Glycoprotease (IMPa). Q: Is it necessary to first remove sialic acids from the substrate prior to digest with O-Glycoprotease (IMPa)? A: No, O-Glycoprotease (IMPa) is fully active on sialylated glycoproteins and glycopeptides. Q: Is O-Glycoprotease (IMPa) active on TF-antigen and Tn-antigen? A: O-Glycoprotease (IMPa) efficiently cleaves peptides containing smaller asialylated glycans such as TF-antigen (Galβ1,3GalNAc-α-) and Tn-antigen (GalNAc-α-). Q: Does O-Glycoprotease (IMPa) require metal ions for activity? A: O-Glycoprotease (IMPa) is a metalloprotease. Treatment with chelating agents such as EDTA inactivates O-Glycoprotease. Addition of Mn2+, Zn2+, Cu2+ will cause decreased enzyme activity.