Serum stability of phosphopeptides

Synthetic phosphopeptides are increasingly used to mimic phosphoprotein fragments and in rational drug design. Both applications require a study of the stability of phosphopeptides in biological environments. Here we report a study of the stability of three sets of synthetic un-, mono-, and diphosphorylated peptides in diluted human serum. The synthetic peptides contained the phosphoserines or phosphothreonines in mid-chain position corresponding to immunodominant regions of abnormally hyperphosphorylated variants of the human τ protein present in the paired helical filaments of Alzheimerʹs disease. The peptide degradation in human serum was monitored, and the first metabolites formed were collected from reversed-phase high performance liquid chromatography (RP-HPLC). Seven of nine monophosphorylated peptides exhibited increased stability compared to the unphosphorylated parent analogs. All peptides phosphorylated at immunodominant sites displayed higher serum stability than phosphopeptide isomers of the same sequences giving evidence of the extended presence of the antigenic stimulus in the hosts. Analysis of the degradation pathway of the fastest degrading peptide family showed that the mono- and diphosphorylated peptides, like the unphosphorylated version, underwent aminopeptidase cleavage and the phosphate group remained attached to the serine and threonine side chains. These results indicate that phosphopeptides are suitable models of phosphoprotein fragments in biochemistry assays, and that phosphorylation can be a viable modification of peptide leads in drug design.