Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence

Analytical Biochemistry
P Fadden, T A Haystead

Abstract

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 ...Continue Reading

Citations

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