Articles tagged ”O-glycans”
OpeRATOR Publication from Johns Hopkins University
Scientists from the prestigious Johns Hopkins University School of Medicine have used OpeRATOR to develop a workflow to map O-glycosylated sites on proteins in very complex samples. O-glycoproteins are notoriously difficult to study due to the low abundance, high structural heterogeneity and low stability. Previous approaches using affinity enrichment or engineered cell culture systems either lack efficiency or are ill-suited forO-glycoproteomic studies of complex samples.
In the workflow developed by Weiming Yang and colleagues, protein samples such as serum or kidney tissue were digested with trypsin, immobilized onto beads through the N-terminus and treated with OpeRATOR and SialEXO. OpeRATOR is an endoprotease and derived from the gut commensal bacteria Akkermansia muciniphila that specifically cleaves peptides and proteins N-terminally of O-glycosylated serine or threonine residues. Therefore, only O-glycopeptides are released from the solid support and were identified using ETD mass spectrometry.
Using this workflow, Yang et al. were able to map over 3000 O-glycosylation sites from human serum, T cells and kidney tissue, almost doubling the number of known O-glycosylation sites. They were also able to detect and quantify the aberrant O-glycosylation patterns in kidney tumors, showcasing the potential use of such methodologies for both basic research and diagnostic purposes.
Meet the Scientist
We got the opportunity to interview the first author of the paper, Weiming Yang at Johns Hopkins University.

For more information on OpeRATOR go the the following pages:
- OpeRATOR Product Page
- Analysis of O-glycosylated proteins
- Download poster on OpeRATOR from ASMS 2018

Poster Presentations at PEGS Europe 2018
This week, scientists from Genovis are presenting two poster at the Protein Engineering Summit in Lisbon, Portugal. The posters cover our O-glycan specific endoprotease Operator and the recently launched FabRICATOR-HPLC column for automated antibidy subunit generation. Check out the poster abstracts below:
An O-glycan Specific Endoprotease with Applications in Glycoprotein Analysis using LC-MS
Helen Nyhlen, Maria Nordgren, Stephan Björk, Rolf Lood, Fredrik Leo, Fredrik Olsson
Genovis AB, Sweden
Changes in protein glycosylation may have an impact on the structure and function of a glycoprotein and O-glycosylation has drawn more and more attention for its roles in a wide range of biological processes. Characterization of glycosylation is of growing importance for the development and quality control of recombinant glycoprotein drugs and biosimilars. The study of O-linked glycosylation within the field of glycoproteomics is however challenging due to complicated sample preparation, difficult analytical procedures and the lack of O-glycan specific enzymes.
An O-glycan specific protease originating from the mucin degrading bacteria Akkermansia muciniphila has been described previously. The enzyme is dependent on the presence of O-glycans for digestion and hydrolyzes the peptide bond N-terminally to O-glycosylated serine and threonine residues. This feature can be used for the generation of intact O-glycopeptides to study site occupancy and composition of O-glycans in various biologic samples. We present here workflows that enabled determination of O-glycan sites and composition for O-glycosylated biopharmaceuticals and for proteins in human serum.
The O-linked glycosylation sites of biopharmaceuticals were assessed by treatment with PNGaseF, sialidases, O-protease and/or trypsin overnight prior LC/MS. The unique MS/MS peptides obtained revealed and defined the O-glycosylated threonine and serine residues. Enrichment of O-glycoproteins from human serum was achieved in native conditions using an affinity binding resin for O-glycan protein based on agarose beads with immobilized inactive O-protease. The complex protein sample was desialylated during the incubation step for binding. Bound proteins were then eluted by urea and treated with PNGaseF, active O-protease and/or trypsin followed by RP-C18 or HILIC separation and ESI-QTOF/MS analysis. The resin displayed high affinity for core 1 mucin-type glycans. With this workflow peptides and O-glycopeptides, with site-specific information, from several serum proteins were identified.
To summarize, using the characteristics of the O-protease and the O-glycoprotein affinity binding resin, strategies for the characterization of O-glycosylated proteins from pure and complex protein samples have been developed. The O-protease and the O-glycoprotein binding resin are potentially useful tools for deep characterization of O-glycoproteins.
Rapid On-column Digestion for Automated Monoclonal Antibody Analysis
Stephan Björk, Andreas Nägeli, Maria Nordgren, Linda Andersson, Helen Nyhlen, Jonathan Sjögren, Fredrik Olsson
Genovis AB, Lund, Sweden
Monoclonal antibodies (mAbs) and other IgG-based biopharmaceuticals are a fast-growing market. The inherent heterogeneity of such biologics necessitates detailed characterization by liquid chromatography and mass spectrometry (LC-MS) during development and production. While bottom-up peptide mapping is still the gold standard for analysis of critical quality attributes, such approaches are resource and time intensive in terms of both data acquisition and analysis. Top-down and middle-down approaches are therefore gaining in popularity. Antibody subunit analysis has become a widely accepted analytical strategy for rapid characterization of therapeutic antibodies and related products. The IdeS enzyme specifically digests IgG just below the hinge, generating F(ab’)2 and Fc/2 fragments. Reduction of disulfide bonds yields fragments of 23-25kDa in size which are amenable to high-resolution mass spectrometry. The IdeS based middle-level LC-MS workflow therefore enables the analysis of multiple antibody quality attributes such as glycosylation, oxidation, and C-terminal lysine clipping.
Here we present a rapid and automatable solution for antibody subunit generation in an HPLC column format. FabRICATOR (IdeS) enzyme was immobilized on the column to allow for automated middle-level analysis in a 2D-HPLC setup. The mAbs are digested on-column in the first dimension and the resulting subunits are separated and analyzed in the second dimension by RP-HPLC. This could be achieved with minor modifications to an HPLC-MS setup and potentially be connected directly to a bioreactor for automated monitoring of an on-going mAb production. The column tolerates continuous operation at 37°C for >10 days without a significant decrease in digestion performance and delivers consistent results for Fc glycan analysis during the entire period of operation. Additionally, other critical quality attributes such as Fab glycosylation and lysine clipping could be monitored. FabRICATOR-HPLC provides a fast solution for antibody subunit generation while reducing sample handling errors and increasing throughput.
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/