OpeRATOR™ for O-glycan site mapping

Application

Precise O-glycan site mapping by generating small, MS-friendly O-glycopeptides and improved confidence through overlapping sequence coverage.

Etanercept is an Fc-fusion protein made up of an Fc-domain from a human IgG1 and the TNFα receptor (TNFR). There are three sites of N-glycosylation on each half of the protein; one on the Fc-domain and two on the TNFR domain, as well as a hinge region which is decorated with up to 13 mucin-type O-glycans on each half of the protein.
Etanercept was digested with FabRICATOR, OpeRATOR and SialEXO in a one-pot-reaction. The FabRICATOR digestion facilitated the O-glycoslylation analysis by separating the TNFR from the Fc domain which led to separation of the most C-terminal O-glycan from the Fc N-glycan. SialEXO was used to remove the sialic acid residues to ensure OpeRATOR activity. The resulting glycopeptides were analyzed using HILIC-LC-MS/MS. A representation of the workflow is shown in Figure 1.
Figure 1. OgpA-based workflow for analysis of the O-glycosylation of etanercept. Etanercept was digested with FabRICATOR, OpeRATOR and SialEXO in a one-pot reaction. The resulting peptdes were reduced and alkylated before analysis by HILIC-LC-MS/MS.

OpeRATOR offers a solution with several advantages over traditional peptide mapping workflows for comprehensive O-glycan site mapping. The etanercept hinge region is highly O-glycosylated and, typically, within these patches of O-glycosylation there are several serine, threonine and proline residues, but not so many lysine or arginine residues. This makes unambiguous glycan site mapping difficult, even when using MS fragmentation techniques such as ETD.

Comprehensive O-glycan site mapping

Use of traditional proteases such as trypsin, often generates very large peptides containing multiple O-glycans and in the case of etanercept, there can be up to seven glycans on a single tryptic peptide. Because OpeRATOR digests at each site of O-glycosylation, when analysing a protein such as etanercept, the high number of O-glycan sites present in close proximity means that the generated peptides are generally small, contain only 1-2 O-glycans per peptide, and are much easier to analyze.

OpeRATOR generates small peptides with typically 1-2 O-glycans per peptide

While OpeRATOR cleaves at each glycan site, some mis-cleavage is also observed. This leads to some peptides containing more than one O-glycan, and also leads to the generation of overlapping peptides. This is useful for glycan site mapping as, while smaller peptides are advantageous for analysis, if they are too small, they would potentially be difficult to identify and possibly impossible to map onto the overall protein sequence, especially within regions which might containing repeating sequences. Overlapping peptides aid unambiguous glycan site identification as there are multiple generated peptides which contain the glycan site which improves the overall confidence in their characterization.

Overlapping peptides increase confidence in glycan site identification

Figure 2. 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.

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