Extraction and purification of indole-3-acetic acid (IAA) and abscisic acid (ABA) metabolites was done as described previously34 (link) with minor modifications. Frozen samples were homogenized using a MixerMill (Retsch GmbH, Haan, Germany) and extracted in 1 ml 50 mM sodium phosphate buffer (pH 7.0) containing 1% sodium diethyldithiocarbamate and stable isotope-labelled internal standards (5 pmol of [13C6]-IAA and [6H2]-ABA per sample added). The pH was adjusted to 2.7 with 1 M hydrochloric acid, and the samples were purified by solid phase extraction. The extracts were purified on Oasis HLB columns (30 mg, Waters Corp., Milford, USA), conditioned with 1 ml methanol, 1 ml water, and 0.5 ml sodium phosphate buffer (pH 2.7). After sample application, the column was washed with 2 ml 5% methanol and then eluted with 2 ml 80% methanol. Eluates were evaporated to dryness and dissolved in 30 ul of mobile phase prior to mass analysis using a 1290 Infinity Binary LC System coupled to the 6490 Triple Quad LC/MS System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies)35 (link).
Dual ion funnel technologies
Manufactured by Agilent Technologies
Sourced in United States
The Dual Ion Funnel technologies at Agilent are designed to efficiently capture, transport, and focus ions in mass spectrometry applications. The dual ion funnel system utilizes two consecutive ion funnels to optimize ion transmission and enhance sensitivity. This technology is intended to improve the overall performance and capabilities of Agilent's mass spectrometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
1290 infinity binary lc system, by Agilent Technologies (2 mentions)
6490 triple quad lc ms system, by Agilent Technologies (2 mentions)
Oasis hlb column, by Waters Corporation (2 mentions)
Jet stream, by Agilent Technologies (2 mentions)
1260 infinity 2 lc system, by Agilent Technologies (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
3 protocols using dual ion funnel technologies
1
Quantitative Analysis of Phytohormone Metabolites
2
Quantifying endogenous ABA in plant roots
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For endogenous ABA quantification, wild-type (cv. Nipponbare) primary root tips (1 cm) were harvested from noncompacted and compacted soil-grown plants. The root tips were gently washed with autoclaved RO water to remove soil particles, immediately snap frozen in liquid nitrogen and stored at −80 °C. Extraction (∼8 to 10 mg fresh weight per sample) was performed according to (36 (link)); cold 10% methanol containing 1 M formic acid (vol/vol) was used as the extraction solvent. In brief, [2H6] ABA was added as an internal standard prior to extraction, and samples were then extracted using 1 mL of extraction solvent, homogenized, and purified using Oasis HLB columns (30 mg/mL, Waters Corporation). For the data in Fig. 1A , ABA measurements were performed by UHPLC-MS/MS using an Acquity UPLC System (Waters) equipped with an Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 µm; Waters) coupled to a triple quadrupole Xevo TQ-S MS (Waters). For SI Appendix, Fig. S1C , analyses were conducted using a Phenomenex Polar C18 column (150 × 2.1 mm, 2.5 µm; Phenomenex), using Milli-Q water (A) and ACN (B), both with 0.02% formic acid (vol/vol) as mobile phase, with measurements performed on a LC-MS/MS system comprising a 1260 Infinity II LC System coupled with 6495 Triple Quad LC/MS System, Jet Stream and Dual Ion Funnel technologies (Agilent Technologies).
3
Extraction and Quantification of Auxin Compounds
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Extraction and purification of the targeted compounds (IAA, oxIAA, IAA-Asp, IAA-Glu, IAAglc, oxIAA-glc) were performed according to (Novak et al., 2012) . Briefly, 10 mg of frozen material per sample was homogenized using a bead mill (27 Hz, 10 min, 4°C; MixerMill, Retsch GmbH, Haan, Germany) and extracted in 1 ml of 50 mM sodium phosphate buffer containing 0.1% sodium diethyldithiocarbamate and a mixture of 13 C6 isotopically labelled internal standards (Olchemim, Olomouc, Czech Republic). After centrifugation (20 000 g, 15 min, 4°C), the supernatant was transferred into new Eppendorf tubes. The pH was then adjusted to 2.5 with 1 M HCl and samples were immediately applied to preconditioned solid-phase extraction columns (Oasis HLB, 30 mg of 1 ml; Waters Inc., Milford, MA, USA). After sample application, each column was rinsed with 2 ml 5% methanol. Compounds of interest were subsequently eluted with 2 ml of 80% methanol. UHPLC-MS/MS analysis was performed according to the method described in (Pencik et al., 2018) , using an LC-MS/MS system consisting of a 1290 Infinity Binary LC System coupled to a 6490 Triple Quad LC/MS System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies, Santa Clara, CA, USA). The quantification was carried out in Agilent MassHunter Workstation Quantitative Analysis software (Agilent Technologies, Santa Clara, CA, USA).
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