In this Technical Note, accuracy, precision, linearity, and limits of detection are documented for a commercially available mix of bovine fetuin N-linked oligosaccharide alditols using an HPAE-PAD method. These results are intended as a guide for investigators who need to validate the performance of HPAE-PAD for oligosaccharide mapping. Readers are also encouraged to see newer AppsLab record for this analysis including AN1050, AU176, and AN215.

Characterization of glycoproteins routinely involves carbohydrate analysis. Studies show that HPAE-PAD is a simple and effective way to determine glycoprotein monosaccharide composition without derivatization. In a typical method, monosaccharides are separated on a Thermo Scientific Dionex CarboPac PA10 or PA20 anion-exchange column, preceded by a Thermo Scientific Dionex AminoTrap guard column, using a hydroxide eluent prepared manually or generated by an eluent generator. This TN reports such a method.

This application note shows how the PA200 improves N-linked oligosaccharide separations. The PA200 is used to profile N-linked oligosaccharides released from human polyclonal IgG by the enzyme PNGase F or endoglycosidase H (Endo H). This note also shows how subsequent exoglycosidase digestions can be used to assist in understanding and identifying oligosaccharide structure.

Glycans released from bovine fetuin by PNGase-F are separated by gradient elution on the Thermo Scientific Dionex GlycanPac AXR-1 column and detected by charged aerosol detection on the Corona Veo RS.

The Thermo Scientific Dionex UltiMate 3000 RSLC system is applied for the analysis of peptide mapping. The separation of four peptide maps was achieved on a Thermo Scientific Acclaim 120 C18 column in 50 minutes with UV detection.