Articles tagged ”Operator”

Mapping the O-glycoproteome Using Site-Specific Extraction of O-linked Glycopeptides

August 20, 2020 | Applications, References |

The Tn-antigen is characterized by the presence of a single N-Acetylgalactosamine (GalNAc) residue linked to serine or threonine residues on proteins. It is an immature glycoform without extension to form any of the common O-glycan core structures. The Tn antigen is a hallmark of many forms of cancer and rarely found in healthy tissue. Mapping and comparing Tn-antigens on proteins is therefore of great interest but current methods are laborious and inefficient. 



Scientists at Johns Hopkins School of Medicine have developed an ingenious new approach for mapping Tn-antigens from a number of complex samples (Yang et al 2020a). It is based on a technique called ExoO (Yang et al 2018) where tryptic peptides are covalently immobilized on a solid support and digested with OpeRATOR®. As this enzyme can only digest when an O-glycan is present, this results in the specific release of O-glycopeptides which can be analyzed in-depth using LC-MS.


However, OpeRATOR does not digest at sites modified with Tn antigens. To circumvent this limitation, Yang et al. first treated their samples with recombinant C1GalT1, an enzyme able to elongate the Tn antigen into a core 1 structure (Gal-β1,3- GalNAc). Sites modified with core 1 O-glycans are efficiently digested by OpeRATOR and can therefore be mapped in depth using EXoO and LC-MS. To distinguish the Tn-antigen sites from naturally occurring core 1 glycosylation sites, the C1GalT1 reaction was performed in the presence of a heavy isotope labelled donor substrate (13C-UDP-Gal). This introduces a unique mass signature that specifically marks the Tn-antigen sites and is easily detectable by mass spectrometry. Using this new approach, termed EXoO-Tn, Yang et al. were able to identify an approximately 10-fold higher number of Tn-glycosylation sites compared to previous studies. 


Link to paper, Yang, W. et al., 2018. Mapping the O-glycoproteome using site-specific extraction of O-linked glycopeptides (EXoO). Molecular systems biology, 14(11), p.e8486. 
For a detailed description on how to use
OpeRATOR for in-depth mapping of O-glycosylation sites using the EXoO workflow, check also this recent publication in Nature Protocols (Yang et al 2020b) 

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.


Weiming Yang

Tell us about yourself?
I am a Research Associate in Mass Spectrometry Core Facility in the Center for Biomarker Discovery and Translation ( of the Johns Hopkins University. The Mass Spectrometry Core Facility carries large-scale proteomics with particular emphasis on protein glycosylation on proteome scale to elucidate functions of glycoproteins on biology and disease. Before this position, I was a postdoc fellow in the same lab and worked on innovative glycoproteomics methods and HIV research. My interest in protein O-linked glycosylation started from every beginning at Hopkins that I was able to identify an O-linked glycosylation site in HIV gp120 from the infectious virion. Later on, I developed a series of glycoproteomic methods to study protein N- and O-linked glycosylation. The development of novel glycoproteomic methodologies led to new areas toward the discovery of the biomarker for HIV reservoir and new insight into cancer biology.


What is new with the ExoO method you have developed?
The major advantage of EXoO is its applicability to analyze clinical samples that is a breakthrough and central to reveal the significance of protein O-linked glycosylation in diseases. Using EXoO, now, scientists can start to gain new insight into their biological systems regarding O-linked glycoproteins. O-linked glycoproteins are ubiquitous on the cell surface and extracellular environment that is highly relevant to new treatment for diseases and diagnostics. Also, the EXoO is advantageous to analyze mucin-type O-linked glycoproteins that cannot be easily analyzed by conventional methods. The EXoO method identifies a large number of O-linked glycosylation sites in the sample that may be easily identified by using other methods such as various enrichments coupled with ETD-MS/MS.


How did you perform the analysis prior to this method?
We tried to use the same solid phase method to immobilize the peptides but released O-glycopeptides using beta-elimination to study site of protein O-linked glycosylation. Beta-elimination is a chemical reaction that can tag the site of protein O-linked glycosylation but give some background release of peptides from the solid support. We tried ETD-MS/MS for O-linked glycopeptide analysis but the number of identification is lower than the use of the current method using EXoO to release the O-glycopeptides.


What are the benefits of applying Operator in the workflow?
The OpeRATOR enzyme is a key component in the workflow. The high specificity of OpeRATOR enabled release of site-specific O-linked glycopeptides from solid phase support. Therefore, the resulting glycopeptides are relatively pure for improved identification.


What can you tell us about what you currently are working on?
Currently, we are applying the method to study different diseases including cancers and HIV reservoir.


How would you describe the impact of OpeRATOR on the O-glycan field?
The discovery of OpeRATOR changes of the game in the field of O-linked glycoproteomics. It makes the analysis of large-scale and site-specific O-linked glycoproteome in clinical samples feasible. For O-glycans, the specificity of OpeRATOR is not completely clear that will need further investigation. O-glycans have many different structures. The glycomic methods may still be the best way to go.


What are your thoughts on the future of O-glycan analysis?
EXoO and OpeRATOR provide unique research tools to identify the site of O-linked glycosylation. So far, the evidence supports that core 1 Gal-GalNAc structure can be studied by the use of OpeRATOR. Glycomic method focus on the identification of all different O-glycan structures with linkage and quantitative information. In the future, the structures of O-linked glycans on the specific sites on the proteins can be revealed in a single workflow.



For more information on OpeRATOR go the the following pages:


The full text paper is available online:

Poster Presentations at PEGS Europe 2018

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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

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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:


Shuang Yang et al., “Deciphering Protein O‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment,” Analytical Chemistry, June 3, 2018, 1–9, doi:10.1021/acs.analchem.8b01834.


More information on OpeRATOR and its applications: