AU176: Preparation of Peptide N-Glycosidase F Digests for HPAE-PAD Analysis

Instrument Type: IC

The work shown here describes glycoprotein deglycosylation with PNGase F, followed by an efficient sample pretreatment using desalting and detergent removal spin columns to remove high concentrations of salt and detergents from small-scale PNGase F digestions. The HPAE-PAD technique was used to separate and detect the oligosaccharides. Oligosaccharides were separated using the Dionex CarboPac PA200 column. The PA200 is a nonporous, high-efficiency, pellicular anion-exchange column that provides high-resolution separations required for oligosaccharide mapping and analysis.

AN1050: Evaluating Protein Glycosylation in Limited-Quantity Samples by HPAE-PAD - Oligosaccharide Method

Instrument Type: IC

By using a combination of acid hydrolysis and enzymatic digestion steps prior to HPAE-PAD analysis, information about a protein's glycosylation, as well as linkages within the glycans, is determined. These methods provide a cost-effective option for investigating glycans without the derivatization required by many other methods. This record details a method for oligosaccharide analysis.

AN105: Glycosylation Analysis of Human Serum Transferrin Glycoforms Using Pellicular Anion-Exchange Chromatography - HPAE-PAD Method

Instrument Type: IC

Once the DNAPac PA100 fractions are obtained, PNGase F digestions can be performed. PNGase F, an amidase, removes N-linked oligosaccharides attached to glycoproteins. The released oligosaccharides from the different PNGase F digested fractions can be analyzed using HPAE-PAD (High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) using a Dionex CarboPac PA100 column. Today we would start with a Dionex CarboPac PA200 column.

AN215: Separation of Asparagine-Linked (N-Linked) Oligosaccharides from Human Polyclonal IgG

Instrument Type: IC

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.