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

Jana Svačinová,Lenka Plačková,Miroslav Strnad,Karel Doležal,Josef Holík,René Lenobel,Ondřej Novák

doi:10.1186/1746-4811-8-17

Abstract

BackgroundWe 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.ResultsPlant 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.ConclusionsThe 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.

Highlights

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
Development of StageTip purification method The optimization of homogenization, extraction and purification, critical and rate-limiting steps in the isolation of natural compounds, is important for small sample sizes, where losses and crosscontamination must be reduced to a minimum
In the highly acidic extraction mixture, cytokinins should be predominantly presented as cations, and they should be completely retained on a cation-exchange sorbent from which they can be eluted with an alkaline solvent

Summary

Introduction

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. For determination of auxin in small samples (0.05–0.1 mg of tissue), a technical level was reached where losses of reproducibility due to factors such as contamination and precision in tissue sampling can pose equal or even larger problems than the analytical sensitivity per se This is still not the case for several other phytohormones, for which improvements in instrumentation and sample purification would greatly facilitate attempts to address questions being asked in developmental biology [18]. StageTips are ordinary pipette tips containing very small disks made of beads with reversed-phase or ion-exchange surfaces embedded in a Teflon mesh [26] These self-made micro-tips combine flexibility, ease and speed of use, small bed volume and high loading capacity, and provide excellent recovery, high reproducibility, robustness and versatility for even minute amounts of analyte. A further advantage for sample preparation is that the one-time usage eliminates the risk of carryover [26]

Objectives

Methods

Results

Conclusion