title: Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry.
abstract: 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 microM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 micromol/L phosphorylated compounds (r(2) = 0.9996), and a relative standard deviation of less than 8% (n = 3) for all MCB responses of the different concentrations of phosphorylated compounds was obtained. The presented method showed specificity for phosphorylated peptides and may prove a useful tool for studying other ligand-exchange reactions and metal-protein interactions.
authors.affiliation: Faculty of Sciences, Division of Chemistry, Department of Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. krabbe@few.vu.nl
authors.forename: J G
authors.initials: JG
authors.lastname: Krabbe
authors.forename: H
authors.initials: H
authors.lastname: Lingeman
authors.forename: W M A
authors.initials: WM
authors.lastname: Niessen
authors.forename: H
authors.initials: H
authors.lastname: Irth
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005240:Feasibility Studies; D008024:Ligands; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D008024:Ligands; D010748:Phosphopeptides