Analysis of O-Linked Glycosylation

O-glycan Site Mapping using OpeRATOR

OpeRATOR is an O-glycan specific protease that digests 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. 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.

Workflow - O-glycan

O-glycan icon

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

SmartEnzymes workflow with O-glycan

Figure 2. OgpA-based workflow for analysis of the O-glycosylation of etanercept.

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, 3) For comparison, a standard tryptic digest of the same material  (Fig 3c), shows only few and very large glycopeptides that are difficult to analyze.


Figure 3. In-depth analysis of the O-glycosylation sites of etanercept originator.
a) HILIC-MS analysis of etanercept peptides generated by digestion with OgpA. BPC (teal) and XIC of the HexNAc oxonium ion at 204.087 (orange) are overlaid.
b) Peptide map of the O-glycosylation sites of etanercept based on the results from the OgpA-based workflow shown above. The identified O-glycosylation sites are marked in yellow. Two sites (marked with an asterisk) could only be inferred from the OgpA digestion pattern without direct identification of a peptide containing the glycosylated amino acid.
c) RP-MS analysis of etanercept digested with trypsin. BPC (teal) and XIC of the HexNAc oxonium ion at 204.087 (orange) are overlaid. Peptides were separated using a Thermo Scientific™ Acclaim™ 120 C18 column (2.2 µm, 2.1 x 150 mm), the rest of the analysis was performed as described above.

Figure 3. In-depth analysis of the O-glycosylation sites of etanercept originator.

O-glycan Site-specific Digestion of EPO using OpeRATOR

With simpler substrate proteins carrying only one or a few O-glycosylation sites OpeRATOR can also be used to map O-glycosylation sites at the middle-level. Erythropoietin (EPO) is a ~30 kDa glycoprotein with a single O-glycosylation site. After removal of N-glycans by PNGase F and desialylation using SialEXO, the native protein was digested with OpeRATOR and the resulting protein fragments analyzed by reverse phase LC-MS. The site of O-glycosylation can be inferred directly from identifying the site of OeRATOR digestion.

Specific Digestion O-glycan

Figure 4. 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.