Glycoproteomic analysis of human fibrinogen reveals novel regions of O-glycosylation.
Human fibrinogen is a 340 kDa, soluble plasma glycoprotein composed of paired sets of three subunits (α, β, γ). The protein plays a crucial role in protecting the vascular network against the loss of blood after tissue injury. The beta and gamma subunits each contain one N-glycosylation site, each of which is occupied by a biantennary N-glycan. So far O-linked oligosaccharides have rarely been described. Here, we make use of tryptic- and proteinase K-generated fibrinogen glycopeptides for the detailed analysis of the protein's O-glycosylation by combining information obtained from both one- and two-dimensional nanoLC-ESI-ion trap (IT)-MS approaches. Glycopeptides were analyzed by ion trap-MS/MS which displayed fragmentations of glycosidic linkages and some peptide backbone cleavages. MS3 spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N-glycan attachment sites of human fibrinogen could be confirmed. Moreover, we describe seven novel O-glycosylation regions in human fibrinogen, all occupied by a monosialylated T-antigen. Our findings may help to improve the general understanding of human fibrinogen in the blood clotting process.
Department of Parasitology, Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, Leiden, The Netherlands. gzauner@lumc.nl
Gerhild
G
Zauner
Marcus
M
Hoffmann
Erdmann
E
Rapp
Carolien A M
CA
Koeleman
Irina
I
Dragan
André M
AM
Deelder
Manfred
M
Wuhrer
Paul J
PJ
Hensbergen
D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000737:chemistry; D005340:Fibrinogen / Q000032:analysis* / Q000737:chemistry; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D036103:Nanotechnology; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D006020:Glycopeptides; D009844:Oligosaccharides; D011134:Polysaccharides; D020543:Proteome; D005340:Fibrinogen; D014357:Trypsin; D019286:Endopeptidase K