Analysis of O-linked Glycosylation

Scientists at Genovis have discovered new O-glycan-specific enzymatic tools including: OpeRATOR® - an O-glycan-specific endoprotease, OglyZOR® - an O-glycosidase, SialEXO® - a sialidase mix for removal of sialic acids. The enzymes allow detailed characterization of O-glycosylation, both in terms of site occupancy and composition determination as demonstrated below.

OpeRATOR Glycosylation

OpeRATOR® Product Page

The OpeRATOR enzyme digests mucin-type O-glycosylated proteins N-terminally of the serine and threonine glycosylation sites

Antibody Characterization

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OpeRATOR Activity

We have discovered an O-glycan-specific endoprotease - OpeRATOR, digesting mucin-type O-glycoproteins N-terminally of the serine and threonine glycosylation sites. This generates glycopeptides carrying O-glycans and enables O-glycan profiling, O-glycopeptide mapping as well as middle level analyses using mass spectrometry. Below you find a schematic overview of the OpeRATOR digestion sites (Fig. 1). O-glycans are required for OpeRATOR activity and the enzyme is not active at N-glycans. With OpeRATOR and SialEXO treatment, the O-glycosylated protein is digested into peptides carrying O-glycans. The digestion occurs N-terminally of the O-glycosylation sites. 

Glycosylation OpeRATOR

Figure 1. Schematic overview of the OpeRATOR activity

LC-MS Analysis of Etanercept using OpeRATOR Maps O-glycan Sites

Etanercept is an Fc-fusion protein with a highly O-glycosylated hinge region. Etanercept was incubated with OpeRATOR and the resulting glycopeptides were analyzed using LC-MS/MS. Due to the heterogeneity in the O-glycan pattern of the protein and the OpeRATOR specificity for O-glycan structures, overlapping peptides were formed that made it possible to acquire a complete map of the O-glycan sites (Fig. 2). 


Figure 2. OpeRATOR digestion of the O-glycosylated hinge region of etanercept. OpeRATOR generates overlapping peptides, making it possible to map the O-glycan sites.

O-glycan Site-specific Digestion of EPO using OpeRATOR

The specificity of OpeRATOR is here demonstrated using erythropoietin (EPO) as a substrate. EPO is a ~30 kDa glycoprotein with a single O-glycosylation site. N-glycans were removed from EPO using PNGaseF and sialic acids were removed using SialEXO. In parallel, OpeRATOR hydrolyzed the protein N-terminally of the serine O-glycan site. After reduction of disulfide bridges with DTT, the resulting two fragments were separated on a RP C4 column and intact mass was analyzed with a Bruker Impact II ESI QTOF MS (Fig. 3).

EPO Glycosylation

Figure 3. Specific digestion N-terminally of the O-glycosylation site. The reduced fragments were separated on a reversed phase C4 column followed by ESI-QTOF MS detection. The EPO protein carrying one core 1 O-glycan was hydrolyzed at a single specific site N-terminally of the O-glycosylated serine.



Protease Specific for O-glycans

The OpeRATOR enzyme digests mucin-type O-glycosylated proteins N-terminally of the glycosylation sites.

  • Digests native mucin-type O-glycosylated proteins
  • 2 h to overnight (16-18 h) reaction
  • Desialylation using SialEXO™ (included) increases performance
  • N-terminally of the mucin-type O-glycosylated serine or threonine



Removal of Sialic Acids from Native Glycoproteins

SialEXO efficiently hydrolyzes all sialic acids from native glycoproteins.

  • Hydrolyzes sialic acids on N- and O-linked glycans
  • 2 h reaction
  • Requires no co-factors
  • α2-3, α2-6 and α2-8-linked sialic acids

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