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Glycoproteomic Analysis of Human Fibrinogen Reveals Novel Regions of O‑Glycosylation
journal contribution
posted on 2012-12-07, 00:00 authored by Gerhild Zauner, Marcus Hoffmann, Erdmann Rapp, Carolien A. M. Koeleman, Irina Dragan, André
M. Deelder, Manfred Wuhrer, Paul J. HensbergenHuman 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.