Title : Site-specific N-glycosylation analysis of human plasma
ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry
Abstract :
- Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism
- In this study, a site-specific glycosylation analysis of human ceruloplasmin ( CP ) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC- ESI-MS/MS)
- A tryptic digest of carboxymethylated CP was subjected to LC- ESI-MS/MS
- Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions , such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide
- Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide
- Of the seven potential N-glycosylation sites , four ( Asn119, Asn339, Asn378, and Asn743 ) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2)
- A small amount of sialylated tetraantennary oligosaccharide was detected
- Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.