Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry.
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.
Harazono
Akira
A
National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, 1-18-1 Kami-yoga, Tokyo 158-8501, Japan. harazono@nihs.go.jp
Kawasaki
Nana
N
Itoh
Satsuki
S
Hashii
Noritaka
N
Ishii-Watabe
Akiko
A
Kawanishi
Toru
T
Hayakawa
Takao
T
D000595:Amino Acid Sequence; D001216:Asparagine / Q000032:analysis* / Q000378:metabolism; D002570:Ceruloplasmin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D010449:Peptide Mapping; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D006020:Glycopeptides; D009844:Oligosaccharides; D001216:Asparagine; D002570:Ceruloplasmin; D014357:Trypsin