Oligosaccharide analyses of glycopeptides of horseradish peroxidase by thermal-assisted partial acid hydrolysis and mass spectrometry Original Research Article

Thermal-assisted partial acid hydrolysis of the carbohydrate moieties of N-glycosylated peptides of horseradish peroxidase (HRP) is used to generate oligosaccharide cleavage ladders. These ladders allow direct reading of components of the oligosaccharides by mass spectrometry. Acid hydrolysis performed with 1.4, 3.1, 4.5, or 6.7 M trifluoroacetic acid at 37, 65, or 95 °C for 30 min to 24 h hydrolyzed mainly the oligosaccharide units of glycopeptides with least peptide bond or amino acid side chain hydrolysis. Tryptic N-glycosylated peptides from HRP with molecular weights of 2533, 2612, 3355, 3673, and 5647 Da were used as test systems in these experiments. Data showed that the most labile group of oligosaccharides is the fucose (Fuc) and the majority of the end cleavage products are peptides with one or no N-acetylglucosamine (GlcNAc) residue linked to Asparagine (Asn). Additionally, the data agree with previous reports that glycopeptides 3355 and 3673 Da carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, glycopeptide 5647 Da carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and glycopeptides 2612 and 2533 Da carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the 2612 Da peptide at Asn286 is partially occupied. This method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.