Simultaneous characterization of sequence polymorphisms, glycosylation and phosphorylation of fibrinogen in a direct analysis by LC-MS.

Fibrinogen is an abundant plasma glycoprotein involved in pathologically important processes like blood clotting, hemostasis and angiogenesis. Sequence polymorphisms and posttranslational modification (PTM) status of fibrinogen are important factors of cardiovascular disease. We aim for the simultaneous analysis of fibrinogen subunits for sequence polymorphisms (SNPs), phosphorylation and glycosylation by top-down mass spectrometry. Fibrinogen was isolated from human plasma of twelve individuals and subunits of fibrinogen were separated by RP-HPLC and subsequently analyzed by high resolution ESI mass spectrometry. Two coding single nucleotide polymorphisms on the Aα- and Bβ-subunit could be identified on the basis of their mass shifts: Three individuals are heterozygous and two are homozygous for Thr312Ala on the Aα-subunit, three individuals are heterozygous for Arg448Lys on the Bβ-subunit. For the Aα-subunit we find mono- and diphosphorylation amounting to about 55% to 71% and O-glycosylation (likely sialyl-T-antigen) from 10% to 17%. N-glycosylation is present with one or two sialic acids in a ratio of about 3:2 and 3:1 for the Bβ and the γ-subunit, respectively. Both SNPs and the PTMs are associated with fibrinogen levels, clotting behavior and thus the risk for cardiovascular diseases. The homozygosity of the SNP at position 312 in the alpha chain for example nearly doubles the risk for ischemic stroke. Isolation and analysis of fibrinogen can be achieved in a few hours from only one drop of blood plasma, and thus the method presented here should assist in a quick assessment and prevention of stroke and infarction.