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1.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1074-1075: 86-90, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29334633

RESUMO

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. The resulting non-fluorous solution containing the nucleotides was then directly injected into an amide-type HILIC column using a mixture of acetonitrile and aqueous ammonium bicarbonate as the mobile phase for gradient elution, and the nucleotides were detected using the negative electrospray ionization MS/MS mode. In this method, the extraction recoveries of the nucleotides ranged from 43.2 to 94.7% within a relative standard deviation of 17%. This method enabled the determination of intracellular concentrations of nucleotides.


Assuntos
Quelantes/química , Cromatografia Líquida/métodos , Fluoretos/química , Nucleotídeos/análise , Nucleotídeos/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células Jurkat , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
2.
Talanta ; 156-157: 1-5, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-27260427

RESUMO

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).


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ensaios Enzimáticos/métodos , Compostos Férricos/química , Iminoácidos/química , Oligopeptídeos/metabolismo , Fosfopeptídeos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/análise , Halogenação , Indicadores e Reagentes , Oligopeptídeos/análise , Oligopeptídeos/isolamento & purificação , Fosfopeptídeos/análise , Fosfopeptídeos/isolamento & purificação , Fosforilação , Rodaminas/análise , Rodaminas/isolamento & purificação , Rodaminas/metabolismo , Espectrometria de Fluorescência/métodos
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