ImpaRATOR™ and OpeRATOR™ – Two Synergistic O-glycoproteases

Application

Combining multiple digestions for O-glycan site mapping with glycan structure analysis for complete characterization of heavily O-glycosylated proteins.

The coverage of O-glycan sites in the heavily glycosylated region of etanercept was analyzed after digestion using ImpaRATOR or OpeRATOR. Peptides corresponding to all 13 O-glycan sites were identified using OpeRATOR, whereas only 10 sites could be unambiguously identified using ImpaRATOR. ImpaRATOR may have a more selective amino acid sequence preference than OpeRATOR (Riley and Bertozzi et al., 2022), here indicated by the lack of digestion at sites S186 and T245.

In addition, ImpaRATOR showed limited cleavage between two adjacent O-glycosylated amino acids, as demonstrated by the lack of peptides from digestion at site S213 and the low intensity of peptides from digestion at sites T200 and T217. The peptides from digestion at site T200 were of higher intensity in the OpeRATOR-digested sample (T184-S199 and T200-H204; Fig. 2band d), whereas the corresponding peptide with a missed cleavage (S184-H204) was of a higher intensity in the ImpaRATOR-digested sample.

OpeRATOR shows a higher O-glycan site coverage

While OpeRATOR showed a higher O-glycan site coverage, digestion with ImpaRATOR enabled characterization of the O-glycan structures, including sialylated species. As an example, the bar graph inserts in the figure show the O-glycan species for the two glycopeptides T184-V198 and T205-P207. This should be compared to digestion with OpeRATOR where the sialic acids had to be removed for enzyme activity, which means that the information about the sialylation is lost.

ImpaRATOR enables characterization of O-glycan structures

Taken together, the data generated from ImpaRATOR and OpeRATOR digestions provide complete information about both the glycan composition and O-glycan sites to allow a more comprehensive characterization of O-glycoproteins.

Riley and Bertozzi, 2022. Deciphering O-glycoprotease substrate preferences with O-Pair. Mol. Omics., Dec 5;18(10):908-922.

ImpaRATOR and OpeRATOR provide synergistic O-glycan site information

Sequence coverage of etanercept after digestion with ImpaRATOR or OpeRATOR. Samples were prepared and data acquired as described in Figure 2. Data was processed in UNIFI (Waters™) using a Peptide Map (Exact Mass MS) method. Graph inserts show the glycan distribution of the two example peptides T184-V198 and T205-P207 after digestion with ImpaRATOR. Sites cleaved only by OpeRATOR are indicated by asterisks.

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