OpeRATOR™ Decodes O-glycans; Publication by FDA and Genovis
Scientist at the Center for Biologics Evaluation and Research, Food and Drug Administration, have, in collaboration with Genovis, developed a method for analyzing O-glycosylated proteins based on a solid phase chemical modification and followed by OpeRATOR digestion. Using this method, up to 8-fold more O-glycosites were discovered as compared to previously reported data.
The method uses an on-bead system to capture tryptic peptides deglycosylated using PNGaseF from a glycoprotein mixture. First, the tryptic peptides are bound via the N-terminus to the beads, and subsequent modifications to the sugars can be carried out. Secondly, the OpeRATOR enzyme is applied to digest the peptide bond, N-terminal of the O-glycosylated serine or threonine. In this way, only O-glycosylated peptides will be cleaved off and enriched. The OpeRATOR digested peptides were then analyzed using LC-MS/MS.
OpeRATOR was launched at the American Society for Mass Spectrometry 2017 and the FDA team quickly became interested in this novel tool. The enzyme originates from Akkermansia muciniphila and has been engineered by Genovis for biotech applications and analytical workflows and denoted OpeRATOR. The enzyme binds to musin type O-glycans and cuts the protein backbone, N-terminally of the O-glycosylated site. OpeRATOR can be used to study site occupancy and composition of O-glycans on biopharmaceuticals and for O-glycomic workflows.
“We establish the method on standard glycoproteins, confirming known O- glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity” (Shuang et al 2018)
The paper has been selected Editor’s choice in Analytical Chemistry and is available using the link below:
More information on OpeRATOR and its applications:
https://www.genovis.com/products/enzymes-for-o-glycans/operator/
