Reversed-phase high-performance liquid chromatographic separation of synthetic phosphopeptide isomers

Selectively phosphorylated synthetic peptides corresponding to the human neurofilament protein middle-sized subunit, H-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly-OH, and its analogues were separated by reversed-phase high-performance liquid chromatography of mixtures consisting of the non-phosphorylated, the diphosphorylated and the two different monophosphorylated isomers. Application of the algorithm for the expected retention times to 4-9 amino acid-long peptide fragments revealed the correct elution order of the monophosphorylated isomers. According to circular dichroism studies, this elution order is also compatible with the possibility of induced conformational orientation on the surface of the bonded phase. Chromatographic analysis of the synthetic phosphorylation reaction indicates that the reaction rates of the two structurally different monophosphorylated peptides are similar, which is in contrast to the in vivo site-directed reaction.