Q exactive mass spectrometer orbitrap detector

For ABA measurements, 50 mg of lyophilized fruit pericarp was ground and resuspended in 80% methanol—1% acetic acid solution containing internal standards (deuterium-labeled hormones; OlChemim Ltd., Olomouc, Czech Republic). The mix was shaken for one hour at 4 °C, and the extracted fraction was incubated overnight at −20 °C. The tissue extractions were conducted in triplicate for each developmental stage. Samples were centrifuged, and the supernatant was vacuum dried and dissolved in 1% acetic acid. A reverse-phase column (OasisHLB) was used, and the eluate was dried and dissolved in a solution of 5% acetonitrile and 1% acetic acid. An autosampler and reverse-phase UHPLC chromatography column, 2.6 μm Accucore RP-MS, 100 mm × 2.1 mm (Thermo Fisher Scientific, San Diego, CA, USA) was used. ABA was separated through a gradient of acetonitrile (2–55%) containing 0.05% acetic acid at a rate of 400 μL/min over 22 min and detected in a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific; San Diego, CA, USA). Targeted Selected Ion Monitoring and Electrospray Ionization in the negative mode were used to detect ABA. Measurements were performed using external calibration curves with the Xcalibur 4.0 and TraceFinder 4.1 SP1.

Four independent biological replicate samples of around 150–200 mg fresh weight of either non-acclimated or cold-acclimated Col-0 and nia1,2noa1–2 seedlings were suspended in 80% methanol-1% acetic acid containing internal standards and mixed by shaking during one hour at 4 °C. The extract was kept a −20 °C overnight, centrifuged, the supernatant dried in a vacuum evaporator, and the dry residue was dissolved in 1% acetic acid and passed through an Oasis HLB (reverse phase) column as described^{51 (link)}. The dried eluate was dissolved in 5% acetonitrile-1% acetic acid, and the hormones were separated using an autosampler and reverse phase UHPLC chromatography (2.6 µm Accucore RP-MS column, 50 mm length x 2.1 mm i.d.; ThermoFisher Scientific) with a 5 to 50% acetonitrile gradient containing 0.05% acetic acid, at 400 µL/min over 14 min.
The phytohormones were analyzed with a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific) by targeted Selected Ion Monitoring (SIM). The concentrations of hormones in the extracts were determined using embedded calibration curves and the Xcalibur 2.2 SP1 build 48 and TraceFinder programs. The internal standard for quantification of ABA was the deuterium-labelled hormone. For JA, dihydrojasmonate (dhJA) was used as internal standard.

The G. rugosa extract was dissolved in 80% methanol-1% acetic acid containing internal standards and mixed by shaking for one hour at 4°C. The extract was stored overnight at -20°C, centrifuged and the supernatant dried in a vacuum evaporator. The dried residue was dissolved in 1% acetic acid and passed through an Oasis HLB (reverse phase) column as described in (Seo et al., 2011 ). To quantify the hormone ABA, the dried eluate was dissolved in 5% acetonitrile-1% acetic acid and the hormone separated using an autosampler and reverse phase UHPLC chromatography (2.6 µm Accucore RP-MS column, 100 mm length x 2.1 mm i.d.; ThermoFisher Scientific) with a 5 to 50% acetonitrile gradient containing 0.05% acetic acid, at 400 µL/min for 21 min. The hormone was analyzed by selected ion monitoring (SIM) with a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific), and its concentration in the extract determined using embedded calibration curves and the Xcalibur 4.0 and TraceFinder 4.1 SP1 programs. The internal standard for quantification was the deuterium-labeled hormone.