Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction

0385991 - ÚEB 2013 RIV NL eng J - Journal Article
Béres, Tibor - Gemrotová, Markéta - Tarkowski, P. - Ganzera, M. - Maier, V. - Fridecký, D. - Dessoy, M. A. - Wessjohann, L. A. - Spíchal, Lukáš - Strnad, Miroslav - Doležal, Karel
Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction.
Analytica Chimica Acta. Roč. 751, Feb (2012), s. 176-181. ISSN 0003-2670. E-ISSN 1873-4324
R&D Projects: GA MŠMT(CZ) LC06034
Grant - others:GA MŠk(CZ) ED0007/01/01; GA ČR(CZ) GA522/08/0920
Program: ED; GA
Institutional research plan: CEZ:AV0Z50380511
Keywords : Cytokinin nucleotides * Capillary electrophoresis * Isopentenyltransferase
Subject RIV: ED - Physiology
Impact factor: 4.387, year: 2012

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 400 000 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 mu mol 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 mu mol 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-QqTOE-MS. Dephosphorylation of ATP was observed as a parallel reaction.
Permanent Link: http://hdl.handle.net/11104/0215206