Q exactive spectrometer orbitrap detector

Aliquots (about 100 mg of fresh weight) of frozen tomato leaves (3 biological replicates) were extracted with 80% methanol −1% acetic acid. Deuterium-labeled hormones [²H₆] ABA and [²H₄] SA, were added as internal standards for ABA and SA quantification, whereas the compound dhJA (dihydrojasmonic acid) was used for JA quantification. For collecting the acid fraction containing SA, ABA, and JA, the extracts passed consecutively through HLB (reverse phase), MCX (cationic exchange), and WAX (ionic exchange) columns (Oasis 30 mg cartridges, Waters, Milford, MA, USA), as described in [59 ].
The final residue was dissolved in 5% acetonitrile −1% acetic acid, and the hormones were separated using a reverse phase (2.6 µm Accucore RP-MS column, 100 mm length x 2.1 mm i.d.) UPLC system (ThermoFisher Scientific) with a 5 to 40% acetonitrile gradient containing 0.05% acetic acid at 0.4 mL/min over 14 min. The hormones were analyzed by electrospray ionization and targeted-SIM using a Q-Exactive spectrometer (Orbitrap detector, ThermoFisher Scientific). The concentrations of hormones in the extracts were determined using embedded calibration curves and the Xcalibur 4.1 SP1 build 48 and TraceFinder 4.0 programs (ThermoFisher Scientific).

Hormones (jasmonic acid, indole-3-acetic acid, isopentenyl adenine, abscisic acid and GA₁₂, GA₁₅, GA₂₄, GA₉, GA₅₁, GA₄, GA₃₄, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₂₉, GA₁, and GA₈ gibberellins) were analyzed in leaves of NT and 35S::AtCDF3 (L 2.3) plants grown under control and salinity conditions for 15 days by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) using a Q-Exactive spectrometer (Orbitrap detector; ThermoFisher Scientific) by the Plant Hormone Quantification Service, IBMCP, Valencia, Spain.

A pool of 100-mg fresh weight of samples was used for each measurement, and divided into three independent biological replicates. Quantification of plant-hormones was performed as described by Reference [73 (link)]. Aliquots of lyophilized material were extracted with 80% methanol-1% acetic acid. Deuterium-labeled hormones (purchased from Prof. L Mander- Canberra, OlChemim Ltd-Olomouc, or Cambridge Isotope Lab- Andover): [17,17-²H]GAn, [²H₅]IAA, and [²H₆]ABA were added as internal standards for quantification of SA and ABA. For quantification of JA, the compound dhJA was used instead. For collecting the acids fraction containing SA, ABA, and JA, the extracts passed consecutively through HLB (reverse phase), MCX (cationic exchange), and WAX (ionic exchange) columns (Oasis 30 mg, Waters). The final residue was dissolved in 5% acetonitrile-1% acetic acid, and the hormones were separated by reverse phase UPHL chromatography (2.6 µm Accucore RP-MS column, 100 mm length × 2.1 mm i.d., ThermoFisher Scientific) with a 5% to 50% acetonitrile gradient. The hormones were analyzed by electrospray ionization and targeted-SIM using a Q-Exactive spectrometer (Orbitrap detector, ThermoFisher Scientific, Spain). The concentrations of hormones in the extracts were determined using embedded calibration curves and the Xcalibur 4.1 SP1 build 48 and TraceFinder programs.