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An Improved in Vivo Deuterium Labeling Method for Measuring the Biosynthetic Rate of Cytokinins
Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic
Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic
Department for Oncology–Pathology, Karolinska Biomics Center, Karolinska Institutet, Z5:02, 17176 Stockholm, Sweden
Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71 Olomouc, Czech Republic
* Author to whom correspondence should be addressed.
Received: 10 October 2010; Accepted: 14 December 2010 / Published: 15 December 2010
Abstract: An improved method for determining the relative biosynthetic rate of isoprenoid cytokinins has been developed. A set of 11 relevant isoprenoid cytokinins, including zeatin isomers, was separated by ultra performance liquid chromatography in less than 6 min. The iP-type cytokinins were observed to give rise to a previously-unknown fragment at m/z 69; we suggest that the diagnostic (204-69) transition can be used to monitor the biosynthetic rate of isopentenyladenine. Furthermore, we found that by treating the cytokinin nucleotides with alkaline phosphatase prior to analysis, the sensitivity of the detection process could be increased. In addition, derivatization (propionylation) improved the ESI-MS response by increasing the analytes' hydrophobicity. Indeed, the ESI-MS response of propionylated isopentenyladenosine was about 34% higher than that of its underivatized counterpart. Moreover, the response of the derivatized zeatin ribosides was about 75% higher than that of underivatized zeatin ribosides. Finally, we created a web-based calculator (IZOTOP) that facilitates MS/MS data processing and offer it freely to the research community.
Keywords: cytokinin; deuterium labelling; biosynthetic rate; UPLC; MS
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Tarkowski, P.; Floková, K.; Václavíková, K.; Jaworek, P.; Raus, M.; Nordström, A.; Novák, O.; Doležal, K.; Šebela, M.; Frébortová, J. An Improved in Vivo Deuterium Labeling Method for Measuring the Biosynthetic Rate of Cytokinins. Molecules 2010, 15, 9214-9229.
Tarkowski P, Floková K, Václavíková K, Jaworek P, Raus M, Nordström A, Novák O, Doležal K, Šebela M, Frébortová J. An Improved in Vivo Deuterium Labeling Method for Measuring the Biosynthetic Rate of Cytokinins. Molecules. 2010; 15(12):9214-9229.
Tarkowski, Petr; Floková, Kristýna; Václavíková, Kateřina; Jaworek, Pavel; Raus, Martin; Nordström, Anders; Novák, Ondřej; Doležal, Karel; Šebela, Marek; Frébortová, Jitka. 2010. "An Improved in Vivo Deuterium Labeling Method for Measuring the Biosynthetic Rate of Cytokinins." Molecules 15, no. 12: 9214-9229.