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

Anal Biochem. 1999 May 15;270(1):9-14. doi: 10.1006/abio.1999.4060.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Chromatography, Affinity / methods*
  • Chromatography, High Pressure Liquid / methods
  • Ferric Compounds*
  • Mass Spectrometry / methods*
  • Phosphopeptides / chemical synthesis
  • Phosphopeptides / isolation & purification*


  • Ferric Compounds
  • Phosphopeptides