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Determination of trehalose-6-phosphate in Arabidopsis thaliana seedlings by hydrophilic-interaction liquid chromatography–mass spectrometry

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Abstract

A hydrophilic-interaction chromatography (HILIC) method coupled to electrospray ionization mass spectrometry (ESI-MS) was developed for the determination of trehalose-6-phophate (Tre6P) in Arabidopsis thaliana seedlings. The method was optimized for MS detection and separation of Tre6P from its isomers, such as sucrose-6-phosphate, by testing eluent pH, type of organic solvent and alkalinizer, and gradient conditions. Tre6P could be resolved from matrix components within 28 min by using a water–acetonitrile gradient (0.2 ml/min) at pH 12 with piperidine as alkalinizer. The method was validated for concentrations between 25 and 4,000 nM Tre6P in A. thaliana seedling extracts. Seedlings were extracted with consecutive liquid-liquid and solid-phase extractions, and analyzed with HILIC-MS. Obtained accuracy (80–120 %) and precision (<24 %) demonstrated the suitability of HILIC-MS for determining Tre6P level variations in plants. The limit of detection (LOD) was 3.5 nM Tre6P in extracts corresponding to 4.1 pmol.g−1 fresh plant weight (FW). This is a considerable improvement with respect to anion-exchange chromatography (AEC)-MS (40 nM) and capillary electrophoresis-MS (80 nM). Furthermore, HILIC-MS analysis times were shorter than with AEC-MS (30 and 60 min, respectively). The applicability of the HILIC-MS method was demonstrated by the analysis of extracts from seedlings grown on medium containing 100 mM sorbitol or trehalose, resulting in mean Tre6P concentrations of 0.2 and 1.9 nmol.g−1 FW, respectively. Similar concentrations were found with AEC-MS. HILIC-MS was also evaluated at a high flow rate (2.0 ml/min). This high-speed method resolved the Suc6P and Tre6P peaks within 3 min yielding a detection limit of 1.3 nM Tre6P.

Extracted-ion chromatogram obtained during high-pressure hydrophillic-interaction liquid chromatography-mass spectrometry of a trehalose-grown Arabidopsis thaliana (photopgraph) seedling. Trehalose-6-phosphate (structural formula) is separated from its isomers sucrose-6-phosphate and lactose-1-phosphate

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Author notes

  1. Thierry L. Delatte

    Present address: Laboratory of Plant Physiology, Wageningen University and Research Centre, P.O. Box 16, 6708PB, Wageningen, The Netherlands

Authors and Affiliations

  1. Biomolecular Analysis, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB, Utrecht, The Netherlands

    Javier Sastre Toraño, Thierry L. Delatte, Gerhardus J. de Jong & Govert W. Somsen

  2. Molecular Plant Physiology, Department of Biology, Utrecht University, P.O. Box 80088, 3508 TB, Utrecht, The Netherlands

    Thierry L. Delatte, Henriette Schluepmann & Sjef C. M. Smeekens

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  1. Javier Sastre Toraño
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  2. Thierry L. Delatte
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  3. Henriette Schluepmann
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  4. Sjef C. M. Smeekens
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  5. Gerhardus J. de Jong
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Correspondence to Javier Sastre Toraño.

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Javier Sastre Toraño and Thierry L. Delatte contributed equally to this work.

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Sastre Toraño, J., Delatte, T.L., Schluepmann, H. et al. Determination of trehalose-6-phosphate in Arabidopsis thaliana seedlings by hydrophilic-interaction liquid chromatography–mass spectrometry. Anal Bioanal Chem 403, 1353–1360 (2012). https://doi.org/10.1007/s00216-012-5928-4

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  • DOI: https://doi.org/10.1007/s00216-012-5928-4

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