Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry

J.G. Krabbe, H. Lingeman, W.M.A. Niessen, H. Irth

Research output: Contribution to JournalArticleAcademicpeer-review


Electrospray ionization mass spectrometry (ESI-MS) is used to selectively detect analytes with a high affinity for metal ions. The detection method is based on the selective monitoring of a competing ligand at its specific m/z value that is released during the ligand-exchange reaction of a metal-ligand complex with analyte(s) eluting from a reversed-phase liquid chromatography column. The ligand-exchange reaction proceeds in a postcolumn reaction detection system placed prior to the inlet of the electrospray MS interface. The feasibility of metal affinity detection by ESI-MS is demonstrated using phosphorylated peptides and iron(III)methylcalcein blue as reactant, as a model system. Methylcalcein blue (MCB) released upon interaction with phosphorylated peptides is detected at m/z 278. The ligand-exchange detection is coupled to a C8 reversed-phase column to separate several nonphosphorylated enkephalins and the phosphorylated peptides pp60 c-src (P) and M2170. Detection limits of 2 μM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 μmol/L phosphorylated compounds (r
Original languageEnglish
Pages (from-to)6853-60
JournalAnalytical Chemistry
Issue number24
Publication statusPublished - 2003


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