Ligand-Exchange Detection of Phosphorylated Peptides Using Liquid Chromatography Electrospray Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 75; no. 24; pp. 6853 - 6860
• Main Authors: Krabbe, J. G, Lingeman, H, Niessen, W. M. A, Irth, H
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.12.2003
• Subjects: Aminoacids, peptides. Hormones. Neuropeptides; Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, Liquid - methods; Exact sciences and technology; Feasibility Studies; Fundamental and applied biological sciences. Psychology; Ligands; Liquids; Other chromatographic methods; Peptides; Phosphopeptides - analysis; Phosphorus; Phosphorylation; Proteins; Sensitivity and Specificity; Spectrometric and optical methods; Spectrometry, Mass, Electrospray Ionization - methods; Spectrum analysis; Complexation; Peptides; Ligand; Liquid chromatography; Exchange reaction; Electrospray; Chemical ionization; Protein; Chemical enrichment; Reversed phase chromatography; Iron III Complexes; Detection limit; Standard deviation; Feasibility; Mass spectrometry; Flow injection
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0349476

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 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.