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Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation

Abstract

The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as theO-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 oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.

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Correspondence to Keith L. Williams.

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Gooley, A.A., Pisano, A., Packer, N.H. et al. Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation. Glycoconjugate J 11, 180–186 (1994). https://doi.org/10.1007/BF00731216

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Keywords

  • N-terminal sequencing
  • glycoprotein
  • glycan analysis
  • covalent immobilization
  • mass spectrometry