Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity

• Published in: Talanta (Oxford) Vol. 156-157; pp. 1 - 5
• Main Authors: Hayama, Tadashi, Kiyokawa, Ena, Yoshida, Hideyuki, Imakyure, Osamu, Yamaguchi, Masatoshi, Nohta, Hitoshi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15-08-2016
• Subjects: Affinity; Chelates; Cyclic AMP-Dependent Protein Kinases - analysis; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Assays - methods; Extraction; Ferric Compounds - chemistry; Fluorescence; Fluorometry measurement; Fluorous; Halogenation; Imino Acids - chemistry; Indicators and Reagents; Kinases; Metal chelate affinity; Oligopeptides - analysis; Oligopeptides - isolation & purification; Oligopeptides - metabolism; Peptides; Phosphopeptide; Phosphopeptides - analysis; Phosphopeptides - isolation & purification; Phosphopeptides - metabolism; Phosphorylation; Protein kinase; Proteins; Rhodamines - analysis; Rhodamines - isolation & purification; Rhodamines - metabolism; Spectrometry, Fluorescence - methods; Substrates; Fluorous; Metal chelate affinity; Protein kinase; Fluorometry measurement; Phosphopeptide
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2016.04.058

We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).
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•A novel fluorous biphasic extraction method was applied to analysis of protein kinase activity.•Fe(III)-immobilized PFIDA was utilized for extraction of phosphopeptides to fluorous phase.•Fluorescent substrate peptide remained in aqueous phase was easily measured.•This method was successfully applied to assay PKA activity.