ImpaRATOR™ Accepts a Wide Range of O-glycan Species

Application

Broad O-glycan substrate acceptance efficiently generating informative O-glycopeptides while retaining glycan structure information.

Etanercept is a fusion protein consisting of the extracellular domain of the TNF receptor linked to the Fc portion of human IgG1. The protein contains a cluster of up to 13 O-glycosylated Ser/Thr residues just above the Fc region, making it a very difficult biopharmaceutical to characterize structurally.

To demonstrate the broad glycan specificity of ImpaRATOR, three samples of etanercept with different glycan compositions were prepared:

  • (i) the untreated glycoprotein carrying a mixture of mono- and di-sialylated core 1 structures
  • (ii) the glycoprotein treated with SialEXO to remove sialic acids and generate naked core 1 structures, and
  • (iii) the glycoprotein treated with SialEXO and GalactEXO to generate single GalNAc residues, also known as Tn antigen.

The three samples were treated with ImpaRATOR and FabRICATOR in a one-pot reaction (Fig. 1). As a comparison, SialEXO-treated etanercept was treated with OpeRATOR and FabRICATOR. The resulting peptides were reduced, alkylated and analyzed by HILIC-MS. FabRICATOR was included in the reaction to separate the Fc region from the most C-terminal glycopeptide to facilitate its characterization. Analysis of the ImpaRATOR-treated samples showed that ImpaRATOR digested all three substrates well and generated similar glycopeptides, but with the expected different glycan species (Fig. 2). The sample treated only with ImpaRATOR contained more variants than the other samples since each peptide was detected with several glycan structures.

ImpaRATOR digestion generates O-glycopeptides while retaining glycan structures

Figure 1. Schematic overview of the digestion workflow using ImpaRATOR. Three samples of etanercept with different glycan compositions were prepared: (i) native etanercept, (ii) etanercept pretreated with SialEXO to remove sialic acids, and (iii) etanercept pretreated with SialEXO and GalactEXO to remove sialic acids and galactose. The three samples were treated with ImpaRATOR and FabRICATOR in a one-pot reaction. The resulting peptides were reduced, alkylated and analysed by HILIC-MS.

The observed glycan species were mainly mono- and di-sialylated core 1 structures, which is in accordance with the expected ones for etanercept. The SialEXO-pretreated samples digested with ImpaRATOR or OpeRATOR both resulted in peptides with naked core 1 structures, with the exception of peptide T205-P207 carrying the Tn antigen observed in the ImpaRATOR-digested sample. Etanercept pretreated with SialEXO and GalactEXO followed by digestion with ImpaRATOR resulted in peptides with only the Tn antigen. Together, this data demonstrates the high glycan substrate acceptance of ImpaRATOR.

ImpaRATOR recognizes a wide range of glycan substrates

The ImpaRATOR-treated etanercept displayed a slightly different digestion pattern than the corresponding desialylated OpeRATOR-treated sample. For example, OpeRATOR digested between two O-glycosylated amino acids more efficiently than ImpaRATOR (for details, see section below). Etanercept with intact sialic acids or the Tn antigen was only digested very weakly by OpeRATOR (data not shown).

Different digestion profiles for ImpaRATOR and OpeRATOR

Figure 2. ImpaRATOR activity on etanercept with different glycan compositions. Etanercept a) untreated, b) treated with SialEXO, or c) treated with SialEXO and GalactEXO was digested by ImpaRATOR and FabRICATOR. d) Etanercept pretreated with SialEXO was digested with OpeRATOR and FabRICATOR. Peptides were reduced, alkylated and separated by HILIC (ACQUITY UPLC Glycoprotein Amide column, 300 Å, 1.7 μm, 2.1 mm × 150 mm) and analyzed on a Waters™ BioAccord™ LC-MS system. Shaded peaks indicate O-glycopeptides shared in (b) and (d).

Related Products

  • ImpaRATOR™ Lyophilized

    Lyophilized enzyme for digestion of mucin-type O-glycoproteins and peptides, including sialylated O-glycan species

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