Fluorous-assisted metal chelate affinity extraction for nucleotides followed by HILIC-MS/MS analysis.

We herein developed a selective method for the determination of nucleotides by fluorous-assisted metal chelate affinity extraction followed by hydrophilic interaction liquid chromatography (HILIC) combined with tandem mass spectrometric (MS/MS) analysis. In this study, the nucleotides were selectively chelated by Fe(III)-immobilized perfluoroalkyliminodiacetic acid, and the resulting chelates were subsequently extracted into a fluorous solvent. The nucleotides present in the fluorous solvent were then back-extracted into a non-fluorous solution, such as a solution of ammonia in aqueous acetonitrile.

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

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).