SEPARATION OF SETS OF MONOPHOSPHORYLATED 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 r e-examined using sets of peptides that included four unphosphorylated, 10 monophosphorylated and 11 diphosphorylated analogs. Trifluoroaceti c acid (TFA) and heptafluorobutyric acid (HFBA) were compared as anion ic ion-pairing reagents in water/acetonitrile gradient runs on a stand ard C-18 analytical column. The separation of the unphosphorylated and monophosphorylated peptides appeared to be dependent upon multiple fa ctors: on-column time, and hydrophilicity of the phosphate group when TFA was used, and general hydrophobicity and partial negative charge w hen HFBA was used. Among the chromatograms developed in the TFA system , increased on-column times resulted in better separation. The retenti on times of some phosphopeptides in which the phosphoamino acids were located proximal to basic residues were reduced compared with other ph osphopeptide isomers in the TFA system, indicating that local in-pepti de ion bridges retain more hydrophilic character of the phosphate side -chain group at pH 1.9. In identical gradients HFBA generally separate d the monophosphorylated peptides from the unphosphorylated parent ana logs 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 b aseline. Remarkably, diphosphorylated peptides frequently eluted as br oad or very broad peaks in both systems, and their retention times wer e sometimes increased compared to the unphosphorylated and monophospho rylated analogs suggesting that the predictive methods for phosphopept ide retention times are not fully applicable for multiphosphorylated s equences. Our data indicate that the RP-HPLC of multiphosphorylated pe ptides and protein fragments need to be further investigated or expand ed with unconventional mobile phases. (C) 1997 Elsevier Science B.V.