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An improved in vivo deuterium labeling method for measuring the biosynthetic rate of cytokinins

P. Tarkowski, K. Floková, K. Václavíková, P. Jaworek, M. Raus, A. Nordström, O. Novák, K. Doležal, M. Sebela, J. Frébortová

. 2010 ; 15 (12) : 9214-9229. [pub] 20101215

Jazyk angličtina Země Švýcarsko

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc12026592

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.

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$a An improved in vivo deuterium labeling method for measuring the biosynthetic rate of cytokinins / $c P. Tarkowski, K. Floková, K. Václavíková, P. Jaworek, M. Raus, A. Nordström, O. Novák, K. Doležal, M. Sebela, J. Frébortová
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$a 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.
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$a Floková, Kristýna $7 _AN069092 $u Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany AS CR & Palacký University, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic
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$a Doležal, Karel, $d 1967- $7 xx0100056 $u Laboratory of Growth Regulators, Palacky University & Institute of Experimental Botany AS CR, Slechtitelů 11, 783 71 Olomouc, Czech Republic. dolezal@risc.upol.cz
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$a Šebela, Marek, $d 1971- $7 jo20000082920 $u Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelů 11, CZ-783 71 Olomouc, Czech Republic. marek.sebela@upol.cz
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