Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence.
Reaction conditions were defined for the selective quantitative derivatization and fluorophore labeling of phosphoserine residues on peptides and proteins. Phosphoserine was derivatized with 1,2-ethanedithiol using a modification of the reaction conditions defined by R. C. Clark and J. Dijkstra (1967) Int. J. Biochem. 11, 577-585 and H. E. Meyer, E. Hoffman-Posorke, H. Korte, and M. G. Heilmeyer (1986) FEBS Lett. 204, 61-66 for stabilizing the phosphoamino acid during Edman degradation reactions. Following derivatization, the thiol-serine residues were coupled to fluorescence by iodoacetate reaction. Characterization by capillary zone electrophoresis and laser-induced fluorescence allowed quantitation of phosphoserine content of peptides and proteins at < 75 amol. In three separate experiments, the overall reaction efficiency for 1,2-ethanedithiol derivatization of phosphoserine was estimated at 89.27 +/- 2.44% (SDM). Subsequent coupling of the derivatized serine residue with 6-iodoacetamidofluoroscein was estimated at > 98% efficiency. Fluorescent probe tagging of phosphoamino acids on proteins and peptides offers direct quantitative evaluation of cellular phosphorylation states at the attomole level in tissue samples derived from plants, animals, and humans, without the use of radioisotopes, antibodies, or mass spectrometry.
Fadden
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Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
Haystead
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TA
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008623:Mercaptoethanol / Q000031:analogs & derivatives; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D010762:Phosphorylase a; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis*; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007461:Iodoacetates; D004676:Myelin Basic Protein; D010455:Peptides; D010748:Phosphopeptides; D011506:Proteins; D011994:Recombinant Proteins; D010768:Phosphoserine; D008623:Mercaptoethanol; C031854:1,2-ethanedithiol; D010762:Phosphorylase a; D019807:Iodoacetic Acid