Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction.
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.
Laboratory of Growth Regulators, Institute of Experimental Botany ASCR, Olomouc, Czech Republic.
Tibor
T
Béres
Markéta
M
Gemrotová
Petr
P
Tarkowski
Markus
M
Ganzera
Vítězslav
V
Maier
David
D
Friedecký
Marco A
MA
Dessoy
Ludger A
LA
Wessjohann
Lukáš
L
Spíchal
Miroslav
M
Strnad
Karel
K
Doležal
D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D003583:Cytokinins / Q000032:analysis* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D057230:Limit of Detection; D013058:Mass Spectrometry; D009711:Nucleotides / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity
D003583:Cytokinins; D009711:Nucleotides; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase