Separation of sets of mono- and diphosphorylated peptides by reversed-phase high performance liquid chromatography

The effect of phosphorylation on the behavior of synthetic peptides in reversed-phase high performance liquid chromatography (RP-HPLC) was re-examined using sets of peptides that included four unphosphorylated, 10 monophosphorylated and 11 diphosphorylated analogs. Trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA) were compared as anionic ion-pairing reagents in water/acetonitrile gradient runs on a standard C18 analytical column. The separation of the unphosphorylated and monophosphorylated peptides appeared to be dependent upon multiple factors: oncolumn time, and hydrophilicity of the phosphate group when TFA was used, and general hydrophobicity and partial negative charge when HFBA was used. Among the chromatograms developed in the TFA system, increased on-column times resulted in better separation. The retention times of some phosphopeptides in which the phosphoamino acids were located proximal to basic residues were reduced compared with other phosphopeptide isomers in the TFA system, indicating that local in-peptide ion bridges retain more hydrophilic character of the phosphate side-chain group at pH 1.9. In identical gradients HFBA generally separated the monophosphorylated peptides from the unphosphorylated parent analogs better than TFA. Nevertheless, most phosphopeptide isomers of the same sequences coeluted in both systems. In identical Chromatographic conditions, TFA produced sharper peaks with a considerably smoother baseline. Remarkably, diphosphorylated peptides frequently eluted as broad or very broad peaks in both systems, and their retention times were sometimes increased compared to the unphosphorylated and monophosphorylated analogs suggesting that the predictive methods for phosphopeptide retention times are not fully applicable for multiphosphorylated sequences. Our data indicate that the RP-HPLC of multiphosphorylated peptides and protein fragments need to be further investigated or expanded with unconventional mobile phases.