Identification and assay of phosphoserine and tyrosine-O-sulphate in fibrinopeptides by reversed-phase high-performance liquid chromatography.

A procedure utilizing reversed-phase high-performance liquid chromatography (HPLC) is described for the identification and quantitation of individual phosphorylated and sulphated fibrinopeptides present in fibrin clot supernatants. Fibrinopeptides from human, rabbit and canine fibrinogens, which have different structures and degrees of phosphorylation and sulphation, were used to demonstrate the applicability of these methods. The procedure relies on the increased peptide hydrophobicity following removal of highly charged phosphate or sulphate groups. Dephosphorylated or desulphated peptides are thus more strongly retained on the reversed-phase HPLC column and are eluted later than their corresponding phosphorylated or sulphated peptide counterparts. Dephosphorylation is achieved by treatment of fibrinopeptide-containing clot supernatants with alkaline phosphatase. Phosphorylated peptides are characterized by an increased retention time resulting from loss of phosphate, whereas non-phosphorylated peptides remain unaffected. Similarly, a prolongation of the peptide retention time resulting from desulphation by mild acid hydrolysis serves to verify sulphation of a peptide.