Seedlings were grown for 7 d in continuous light. The ground tissue (about 150 mg of frozen seedlings) was resuspended in 80% (v/v) methanol-1% (v/v) acetic acid containing the internal standard deuterium-labeled hormone (2H6-ABA) and mixed by shaking during 1 h at 4°C. The extract was kept at −20°C overnight. After centrifugation, the supernatant was dried in a vacuum evaporator. The dry residue was dissolved in 1% (v/v) acetic acid and passed through a reverse phase Oasis HLB column. The final residues were dissolved in 5% (v/v) acetonitrile-1% (v/v) acetic acid. ABA hormone was then separated using an autosampler and reverse-phase Ultra High Performance Liquid Chromatography (2.6 µm Accucore RP-MS column, 50 mm length × 2.1 mm i.d.; ThermoFisher Scientific) with a 5%–50% (v/v) acetonitrile gradient containing 0.05% (v/v) acetic acid, at 400 µL·min−1 over 14 min. The ABA was analyzed with a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific) by targeted selected ion monitoring. The concentrations of ABA in the extracts were determined using embedded calibration curves and the Xcalibur 4.0 and TraceFinder 4.1 SP1 programs.
Orbitrap detector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Orbitrap detector is an advanced mass spectrometry instrument that utilizes an orbital trapping mechanism to precisely measure the mass-to-charge ratio of ionized molecules. It offers high-resolution, accurate mass measurements, and can be used for a variety of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Accucore rp ms column, by Thermo Fisher Scientific (3 mentions)
Dionex ultimate 3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Accucore rp ms, by Thermo Fisher Scientific (1 mentions)
Deuterium labeled hormones, by OlChemIm (1 mentions)
8 protocols using orbitrap detector
1
Quantitative Determination of ABA
2
Identification of Organic Compounds by UHPLC-HRMS
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The instrumental analysis used to identify organic compounds in the EMCHS extract was developed using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Sunnyvale, CA, USA) coupled to an HRMS mass spectrometer (Q Exactive focus with an Orbitrap® detector, Thermo Scientific). A Hypersil GOLD TM column 100 × 2.1 mm, 3 µm was used as stationary phase. The sample was eluted using a mobile phase formed by solvent A (H2O acidified with 0.1% formic acid) and solvent B (acetonitrile acidified with 0.1% formic acid), with a flow rate of 0.1 mL/min. The oven temperature was 40 °C, and 10 µL of the sample was injected. The detection was carried out in electrospray positive ion mode [(+) ESI], recorded in full scan mode. Scan range: (250–700 m/z); microscan: 1 scan/sec. The ESI conditions were as follows: sheath gas flow rate: 30, aux gas flow rate: 10, sweep gas flow rate: 0, spray voltage: 3.0 kV, capillary temperature: 325 °C, S-lens RF level: 100, aux gas heater temperature: 250 °C. Samples were measured in full scan mode, and later, the data were acquired in Selected Ion Monitoring (SIM) and data-dependent (ddMS2) acquisition mode. Confirmation for the m/z 617.4380 and m/z 619.4538 was carried out. The performance of fragmentation was at CE 35 eV and 50 eV, respectively.
3
Identification of Organic Compounds by UHPLC-HRMS
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The instrumental analysis used to identify organic compounds in the EMCHS extract was developed using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Sunnyvale, CA, USA) coupled to an HRMS mass spectrometer (Q Exactive focus with an Orbitrap® detector, Thermo Scientific). A Hypersil GOLD TM column 100 × 2.1 mm, 3 µm was used as stationary phase. The sample was eluted using a mobile phase formed by solvent A (H2O acidified with 0.1% formic acid) and solvent B (acetonitrile acidified with 0.1% formic acid), with a flow rate of 0.1 mL/min. The oven temperature was 40 °C, and 10 µL of the sample was injected. The detection was carried out in electrospray positive ion mode [(+) ESI], recorded in full scan mode. Scan range: (250–700 m/z); microscan: 1 scan/sec. The ESI conditions were as follows: sheath gas flow rate: 30, aux gas flow rate: 10, sweep gas flow rate: 0, spray voltage: 3.0 kV, capillary temperature: 325 °C, S-lens RF level: 100, aux gas heater temperature: 250 °C. Samples were measured in full scan mode, and later, the data were acquired in Selected Ion Monitoring (SIM) and data-dependent (ddMS2) acquisition mode. Confirmation for the m/z 617.4380 and m/z 619.4538 was carried out. The performance of fragmentation was at CE 35 eV and 50 eV, respectively.
4
Quantifying Plant Hormone Levels in Seedlings
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Four independent biological replicate samples of around 150–200 mg fresh weight of either non-acclimated or cold-acclimated Col-0 and nia1nia2noa1-2 whole seedlings were suspended in 80% methanol–1% acetic acid containing internal standards and mixed by shaking for 1 h 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 in Seo et al. (2011) (link). The dried eluate was dissolved in 5% acetonitrile–1% acetic acid, and the hormones were separated using an autosampler and reversed phase UHPLC chromatography (2.6 µm Accucore RP-MS column, 50 mm length×2.1 mm i.d., Thermo Fisher Scientific) with a 5–50% acetonitrile gradient containing 0.05% acetic acid, at 400 µl min−1 over 14 min. The phytohormones were analysed with a Q-Exactive mass spectrometer (Orbitrap detector, Thermo Fisher Scientific) by targeted selected ion monitoring. 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 standards for quantification of each of the different plant hormones were the deuterium-labelled hormones.
5
HPLC-MS Analysis of Compound Separation
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The measurements were carried in a Jasco HPLC-DAD model MD-2015 plus, controlled with the chrompass software. An InertSustain C18 column 250 × 4.6 mm, 3 µm, was used as a stationary phase.
The correct order of elution was confirmed by MS. First, the sample was separated using HPLC-DAD, and full fraction collected was measured by direct injection in mass spectrometer Q Exactive focus with an Orbitrap® detector (Thermo Scientific).
The correct order of elution was confirmed by MS. First, the sample was separated using HPLC-DAD, and full fraction collected was measured by direct injection in mass spectrometer Q Exactive focus with an Orbitrap® detector (Thermo Scientific).
6
Quantifying Hormones in Immature Fruits
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Hormones (indole-3-acetic acid, and GA1 and GA4 gibberellins) were analysed in the fruits (17 days after anthesis (DAA) immature green fruits; approx. 2 cm diameter) by liquid chromatography-electrospray ionisation-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. Four independent extractions were performed for each genotype. The fruit pericarp of three different plants (2 fruits per plant) were pooled in each biological replicate.
7
Quantification of Plant Hormones
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ABA, IAA, GA1, and GA4 were measured in both varieties at 34, 38, and 44 DAFB in the 2018–2019 season. For the extraction, 10 mg of flesh- and peel-enriched tissue was freeze-dried, ground, and suspended 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 4ºC, and the extracted fraction was maintained at − 20ºC overnight. The samples were centrifuged, and the supernatant was vacuum dried and then dissolved in 1% acetic acid. A reverse-phase column (OasisHLB) was used58 , and the eluate was dried and dissolved in 5% acetonitrile—1% acetic acid. An autosampler and reverse-phase UHPLC chromatography column, 2.6 µm Accucore RP-MS, 100 mm × 2.1 mm (ThermoFisher Scientific, San Diego, CA, USA) were used. Then the hormones were separated using a gradient of acetonitrile (2%-55%) containing 0.05% acetic acid, at a rate of 400 µL/min over 22 min. ABA, IAA, GA1, and GA4 were 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 the hormones58 . The quantifications were performed using external calibration curves with the Xcalibur 4.0 and TraceFinder 4.1 SP1.
8
Quantification of Phytohormones in Plants
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Determination of free 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ET, was performed as described by Bulens et al. (2011) with minor modifications: 0.5 g of fresh weight were extracted in 4 mL of 5% sulfosalicylic acid and ACC content was estimated indirectly by measuring the ethylene liberated from extracts after treatment with NaOCl in the presence of Hg 2+ . Spikes of defined amounts of ACC were added for reaction efficiency normalization, and a calibration curve of diluted pure ethylene was employed for ACC quantification. GAs, ABA and JA were extracted and purified following the method of Seo et al. (2011) . Then, 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). Separated hormones were analyzed in a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific) by targeted Selected Ion Monitoring (SIM), and their concentrations in the extracts determined using embedded calibration curves and the Xcalibur 2.2 SP1 build 48 and TraceFinder programs. Deuterium-labeled hormones were utilized as internal standards for quantification, except in the case of JA, for which we used dhJA. In all cases, hormone measurements were performed in quintuplicate employing fully independent tissue samples.
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