Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction
Language English Country Netherlands Media print-electronic
Document type Journal Article
PubMed
23084068
DOI
10.1016/j.aca.2012.08.049
PII: S0003-2670(12)01279-2
Knihovny.cz E-resources
- MeSH
- Alkyl and Aryl Transferases metabolism MeSH
- Arabidopsis enzymology MeSH
- Cytokinins analysis metabolism MeSH
- Electrophoresis, Capillary methods MeSH
- Enzyme Assays methods MeSH
- Mass Spectrometry MeSH
- Limit of Detection MeSH
- Nucleotides analysis metabolism MeSH
- Recombinant Proteins metabolism MeSH
- Sensitivity and Specificity MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- adenylate isopentenyltransferase MeSH Browser
- Alkyl and Aryl Transferases MeSH
- Cytokinins MeSH
- Nucleotides MeSH
- Recombinant Proteins MeSH
A capillary zone electrophoresis (CZE) method for separation of adenosine and N(6)-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69-1.27 μmol L(-1)). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R(2)>0.999) was achieved over the concentration range 5-1000 μmol L(-1). The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE-ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products - isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.
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