CHARACTERIZATION OF A SINGLE GLYCOSYLATED ASPARAGINE SITE ON A GLYCOPEPTIDE USING SOLID-PHASE EDMAN DEGRADATION

The characterization of site-specific glycosylation is traditionally d ependent on the availability of suitable proteolytic cleavage sites be tween each glycosylated residue, so that peptides containing individua l glycosylation sites are recovered. In the case of heavily glycosylat ed domains such as the O-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharid es attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered aft er sequential solid-phase Edman degradation, subjected to acid hydroly sis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) de rivative was further characterized by ionspray mass spectrometry. Comp arison between an endoproteinase Glu-C glycopeptide and a tryptic glyc opeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.