Isolation and comparative peptide mapping of fibrinogen subunits by reversed-phase high-performance liquid chromatography

Abstract

Comparative peptide mapping represents one approach to identification of structural defects in variant human fibrinogens. In view of the large size of this protein, we chose to generate peptide maps of fibrinogen subunits. A reversed-phase high-performance liquid chromatographic method was developed to isolate the subunits: fibrinogen was reduced with dithioerythritol and alkylated with iodoacetamide. Subunits were isolated on a Vydac TP, C 4 column (25 × 1.0 cm). Eluent A was 0.1% aqueous trifluoroacetic acid (TFA); eluent B was 0.1% TFA in acetonitrile. Initial conditions were 65% A, 35% B, at 2 ml/min. The reduced-alkylated subunits were lyophilized, redissolved in 0.1% TFA plus 4–8 M guanidine-HCl, and chromatographed using a linear gradient (1%/min) to 50% B. This procedure provides homogeneous subunits in yields exceeding 90%, and is therefore superior to conventional cation-exchange chromatography. For comparative peptide mapping, the same stationar and mobile phases were used, except that the initial conditions were 90% A/10% B, and a linear gradient to 60% B (1%/min) was used. Alternatively, peptide maps were generated with a 10 × 0.46 cm Spherisorb ODS-2 column and very shallow gradients. The mapping procedure resolves 45–60 peptides with excellent reproducibility, and has been applied to the identification of an apparent polymorphism in fibrinogen Baltimore II, and the structural defect in fibrinogen Baltimore IV.