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

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Fadden, P; Haystead, TA

Published Date

  • February 10, 1995

Published In

Volume / Issue

  • 225 / 1

Start / End Page

  • 81 - 88

PubMed ID

  • 7539987

Pubmed Central ID

  • 7539987

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1006/abio.1995.1111

Language

  • eng

Conference Location

  • United States