We demonstrate a workflow combining chemical release of O-linked glycans, followed by sample cleanup and analysis by HPAE-PAD hyphenated to a Q Exactive HF Hybrid Quadrupole-Orbitrap MS. A recently introduced Dionex CarboPac PA300-4μm column, enables simultaneous separation of neutral and charged glycans without the need for derivatization. High-resolution MS data and MS/MS spectra with diagnostic fragment ions provide reliable structural annotations of heterogeneous glycans. We applied HPAE-PAD/MS to analyze O-glycan structures released from four different glycoproteins.

Glycoprotein characterization and glycosylation profiling are important tasks in the development and production of biopharmaceutical proteins. This application note demonstrates Dual EGC capability and performance for profiling N-linked oligosaccharides released from glycoproteins using high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD). This record shows the separation of N-linked glycans from human acid-1 glycoprotein.

Glycoprotein characterization and glycosylation profiling are important tasks in the development and production of biopharmaceutical proteins. This application note demonstrates Dual EGC capability and performance for profiling N-linked oligosaccharides released from glycoproteins using high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD). This record shows the separation for IgG N-linked glycans.

This application update demonstrates improved resolution of sialylated N-glycans on Thermo Scientific™ Dionex™ CarboPac™ PA200 columns. Starting with a recently described method, changes to elution conditions were tested to improve resolution of sialylated N-glycans enzymatically released from four different glycoproteins. Separations were first evaluated on the analytical format (3 × 250 mm column). Next, the possibility that a shorter column, such as a guard column, would allow significantly improved throughput was evaluated. Finally, a narrow bore format 1 × 250 mm column was tested.

This study demonstrates use of a commercially available glycopeptide standard as a control to determine glycoprotein hydrolysis reaction efficiency. Because the monosaccharide composition of glycopeptide is known, hydrolysis efficiency under a set of conditions can be easily calculated. The monosaccharide yield for the glycopeptide was compared with that of two glycoproteins under identical conditions. This comparison revealed efficient hydrolysis using the conditions tested here. Minimally the glycopeptide standard serves as a positive control for glycoprotein monosaccharide determination.