NEW! Sugar-free Proteins in a Few Hours using OmniGLYZOR

OmniGLYZOR hydrolyzes N- and mucin-type O-glycans. It contains a mixture of immobilized enzymes for fast and efficient removal of N- and simple mucin-type O-glycans on antibodies, fusion proteins and other glycosylated proteins.

Removal of glycans is widely used to reduce heterogeneity to facilitate analysis of the protein by for example mass spectrometry. Deglycosylation can also be used to study the functional role of the glycans.
 

OmniGLYZOR contains a mixture of immobilized enzymes able to sequentially break down the O-glycans for the complete removal of the most commonly occurring O-glycan structures (di- and mono-sialyl core 1 and Tn antigen). We demonstrate the performance of OmniGLYZOR using two therapeutic proteins as substrates, the heavily glycosylated Fc-fusion protein etanercept and erythropoietin (EPO). Due to the glycan heterogeneity, intact analyses resulted in complex mass spectra. By incubating the samples with OmniGLYZOR for 1 hour at 37°C, the N-and O-glycans were efficiently removed as indicated by single peaks corresponding to the unmodified proteins. For EPO, a minor amount of O-glycans modified with acetylated sialic acids were left on the protein since those structures were inefficiently hydrolyzed by OmniGLYZOR. Both substrate proteins were also treated with another commercially available deglycosylation product according to the manufacturer’s recommendation (O/N incubation at 37°C), and the data are shown for comparison.

 
Complete hydrolysis of N- and O-glycans by OmniGLYZOR. Deglycosylation of etanercept (left) and EPO (right). The substrate proteins were incubated on OmniGLYZOR Microspin columns for 1 h at 37°C, or with another commercially available deglycosylation product for 1 h and 20 h at 37°C. To simplify the analysis of etanercept, the deglycosylated protein was digested with FabRICATOR to separate the O-glycosylated TNFR domain from the Fc fragment. The resulting subunits were analyzed by reversed-phase LC-MS using a Waters™ BioAccord™ LC-MS system. EPO was analyzed in the same way in its intact state. The peaks corresponding to the fully deglycosylated substrate proteins are shaded in orange.

OmniGLYZOR – Deglycosylates 5 × 50-100 µg or 10 × 50-100 µg glycoprotein.