Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry.
A method for the detection of trehalose-6-phosphate (T6P) in tissue of the model plant Arabidopsis thaliana is presented. Liquid-liquid extraction (LLE) and mixed mode solid-phase extraction (SPE) were used for sample pretreatment followed by anion exchange chromatography (AEC) coupled with electrospray ionization mass spectrometry (MS) for highly selective quantitative analysis. LLE of plant material was performed with chloroform/acetonitrile/water (3:7:16, v/v/v) followed by SPE with Oasis MAX material, which significantly reduced the complexity of the extracts. On-line coupling of MS with gradient AEC using a sodium hydroxide eluent was accomplished with a postcolumn ion suppressor. The method allows specific quantification of T6P with good linearity for spiked plant extracts, from 80 nM to 1.3 microM (r(2)>0.98). The limit of detection in plant extracts was 40 nM. The recovery of the method was above 80% for relevant T6P levels. The method was applied to the determination of T6P in seedlings from four mutant A. thaliana lines (TRR1-4) resisting growth arrest caused by external supply of trehalose. Results reveal that T6P accumulation differed substantially in the four mutant lines and wild type (WT). It is concluded that the mutants circumvent the growth arrest observed in WT seedlings on 100mM trehalose by different mechanisms.
Delatte
Thierry L
TL
Department of Biomedical Analysis, Utrecht University, 3584 CA Utrecht, The Netherlands. t.l.delatte@uu.nl
Selman
Maurice H J
MH
Schluepmann
Henriette
H
Somsen
Govert W
GW
Smeekens
Sjef C M
SC
de Jong
Gerhardus J
GJ
D000837:Anion Exchange Resins; D017360:Arabidopsis / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D036226:Seedlings / Q000737:chemistry*; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013403:Sugar Phosphates / Q000737:chemistry* / Q000302:isolation & purification*; D014199:Trehalose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification
D000837:Anion Exchange Resins; D013403:Sugar Phosphates; C082722:trehalose-6-phosphate; D014199:Trehalose