A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction

0385990 - ÚEB 2013 RIV GB eng J - Článek v odborném periodiku
Svačinová, Jana - Novák, Ondřej - Plačková, Lenka - Lenobel, René - Holík, Josef - Strnad, Miroslav - Doležal, Karel
A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction.
Plant Methods. Roč. 8, _ (2012), s. 17. E-ISSN 1746-4811
Grant CEP: GA TA ČR TA01010861; GA AV ČR KAN200380801
Grant ostatní: GA MŠk(CZ) ED0007/01/01
Program: ED
Výzkumný záměr: CEZ:AV0Z50380511
Klíčová slova: Pipette tip solid-phase extraction (PT-SPE) * Arabidopsis thaliana * Cytokinins
Kód oboru RIV: EC - Imunologie
Impakt faktor: 2.667, rok: 2012

Background: We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high performance liquid chromatography-fast scanning tandem mass spectrometry. Results: Plant tissue samples (1-5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2- microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. Conclusions: The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.
Trvalý link: http://hdl.handle.net/11104/0220064