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 (198...

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Bibliographic Details
Published in:Analytical biochemistry 1995-02, Vol.225 (1), p.81-88
Main Authors: Fadden, P., Haystead, T.A.J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Chromatography, High Pressure Liquid
Escherichia coli
Fluorescent Dyes
Indicators and Reagents
Iodoacetates
Iodoacetic Acid
Mercaptoethanol - analogs & derivatives
Microchemistry - methods
Molecular Sequence Data
Myelin Basic Protein - chemistry
Peptides - chemistry
Phosphopeptides - chemistry
Phosphopeptides - isolation & purification
Phosphorylase a
Phosphorylation
Phosphoserine - analysis
Proteins - chemistry
Recombinant Proteins - chemistry
Sensitivity and Specificity
Spectrometry, Fluorescence - methods
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Summary: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 (1987) Int. J. Biochem. 11, 577-585 and H. E. Meyer, E. Hoffmann-Posorke, H. Korte, and M. G. Heilmeyer (1986) FEBS Lett. 204, 81-66 for stabilizing the phosphoamino acid during Edman degradation reactions. Following derivatization, the thiol-serine residues were coupled to fluorescene by iodoacetate reaction. Characterization by capillary zone electrophoresis and laser-induced fluorescence allowed quantitation of phosphoserine content of peptides and proteins 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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1995.1111