Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides.

A method based upon immobilized metal ion affinity chromatography (IMAC) is described for purification of phosphopeptides from the crude preparations of solid-phase peptide synthesis step. Affinity chromatography consists of iron(III) immobilized on iminodiacetate-agarose gel. The method was applied for purification of seven synthetic enkephalin-related phosphorylated peptides. The effectiveness of the method was evaluated by analyzing the IMAC-retained and -nonretained components using reversed-phase (RP) high-performance liquid chromatography (HPLC) and an on-line combination of RP-HPLC and electrospray ionization mass spectrometry. The UV and total ion current chromatograms demonstrated that the phosphopeptides were effectively separated and purified.