Quantification of auxin metabolites was performed according to the method described by Novák et al. [60 (link)]. Approximately 10 mg of root or shoot tissue were homogenized and extracted with 1 mL of cold 50 mM sodium-phosphate buffer (pH 7.0) containing 0.1% sodium diethyldithiocarbamate and mixture of internal standards containing 5 pmol of [2H4]ANT, [2H5]IAM, [2H4]IPyA, [13C6]IAA, [13C6]oxIAA, [13C6]IAA-Asp, [13C6]IAA-Glu, [13C6]IAA-Glc, [13C6]oxIAA-Glc and 25 pmol of [2H5]Trp and [2H4] IAN. After centrifugation at 36000 g for 10 min, one-half of each sample was acidified with 1 M HCl to pH 2.7 and purified by solid-phase extraction (SPE) using the Oasis™ HLB columns (30 mg, 1 mL; Waters, Milford, MS, USA). For quantification of IPyA, the second half of the sample was derivatized with cysteamine (0.25 M, pH 8.0) for 1 h, acidified with 3 M HCl to pH 2.7, and purified by SPE. After evaporation under reduced pressure, the auxin content of the samples was analyzed using the 1260 Infinity II HPLC system (Agilent Technologies, CA, USA) equipped with a Kinetex C18 (50 mm × 2.1 mm, 1.7 μm; Phenomenex). The LC system was linked to a 6495 Triple Quad Detector (Agilent Technologies, USA).
6495 triple quad detector
Manufactured by Agilent Technologies
Sourced in United States
The 6495 Triple Quad detector is a high-performance liquid chromatography mass spectrometry (LC-MS/MS) system designed for quantitative and qualitative analysis. It features a triple quadrupole mass analyzer, providing accurate and sensitive detection of target analytes.
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Lab products found in correlation
1260 infinity 2, by Agilent Technologies (3 mentions)
Kinetex c18 column, by Phenomenex (3 mentions)
1260 infinity 2 hplc system, by Agilent Technologies (2 mentions)
Oasis hlb column, by Waters Corporation (2 mentions)
Kinetex c18, by Phenomenex (1 mentions)
Bond elut, by Agilent Technologies (1 mentions)
5 protocols using 6495 triple quad detector
1
Quantification of Auxin Metabolites
2
Quantifying Indole-3-Acetic Acid Metabolites
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Determination of indole-3-acetic acid (IAA) metabolite levels was performed following the methods described before [83 (link)]. As tissue, five-day-old dark-grown hypocotyls of pER8:YUC6 [75 (link)], pMDC7::GH3.6 and pMDC7 empty vector control (EV) lines, induced for 3 h on 2 µM β-Estradiol were used. Briefly, 10 mg of tissue were extracted with 1 mL of 50 mM phosphate buffer (pH 7.0) containing 0.1% sodium diethyldithiocarbamate and mixture of stable isotope-labeled auxins standards. A 200 µL portion of each extract was acidified with 1 M HCl to pH 2.7 and purified by in-tip micro solid phase extraction. After evaporation under reduced pressure, samples were analyzed using HPLC system 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) equipped with Kinetex C18 column (50 mm × 2.1 mm, 1.7 µm; Phenomenex, Torrance, CA, USA). The LC system was linked to 6495 Triple Quad detector (Agilent Technologies, Santa Clara, CA, USA). All samples were measured in quadruplicate for each genotype.
3
Quantification of IAA and oxIAA in Tomato
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Extraction of indole-3-acetic acid (IAA) and 2-oxindole-3-acetic acid (oxIAA) was performed as described in detail in Pěnčík at al. [82 (link)]. Briefly, tomato leaf samples (10 milligrams of fresh weight) were homogenized and extracted in 1 mL of 50 mM sodium phosphate buffer (pH 7.0) with the addition of internal standards: [indole-13C6]IAA and [indole-13C6]oxIAA. The samples were incubated at 4 °C with continuous shaking and then centrifuged (15 min, 23,000× g at 4 °C). Supernatants were then acidified with 1 M HCl to pH 2.7 and purified by solid phase extraction (SPE) using C8 columns (Bond Elut, 500 mg, 3 mL; Varian). After evaporation under reduced pressure, samples were analysed using HPLC system 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) equipped with Kinetex C18 column (50 mm × 2.1 mm, 1.7 µm; Phenomenex) and linked to 6495 Triple Quad detector (Agilent Technologies, Santa Clara, CA, USA) following the methodology described in Novák et al. [83 (link)].
4
Auxin Quantification in Leaf Tissue
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For auxin measurements, 100 mg fresh weight of leaf tissue (leaves 8–12) per sample was collected and shock-frozen in liquid nitrogen under white light (time points during light exposure) or green safety light (time points during night). Analysis was carried out using high-performance liquid chromatography–electrospray tandem mass spectrometry as described in [67 (link)] using 10 mg tissue per biological replicate. Samples were extracted with 1 mL of 50 mM phosphate buffer (pH 7.0) containing 0.1% sodium diethyldithiocarbamate. [13C6]IAA, [13C6]oxIAA, [13C6]IAA-Glc, [13C6]oxIAA-Glc, [13C6]IAA-Asp and [13C6]IAA-Glu (5 pmol of each) were added as internal standards. A 200 µL portion of extract was acidified with 1M HCl to pH 2.7 and purified by in-tip micro solid-phase extraction (in-tip µSPE). After evaporation under reduced pressure, samples were analyzed using HPLC system 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) equipped with Kinetex C18 column (50 mm × 2.1 mm, 1.7 µm; Phenomenex) and linked to 6495 Triple Quad detector (Agilent Technologies, Santa Clara, CA, USA). Auxin levels were quantified using stable-isotope-labeled internal standards as a reference.
5
Auxin Metabolite Analysis by LC-MS/MS
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The evaporated samples processed by in-tip µSPE were dissolved in 30 µL of 10% methanol. Samples processed by SPE on Oasis™ HLB columns (Waters Corp., Milford, CT, USA) were dissolved in 40 µL of 10% methanol. All samples were mixed, sonicated for 5 min, and filtered using a Micro-spin® filter tube (0.2 μm pore size; 3 min at 8000 rpm, (Chromservis, Praha, Czech republic). Determination of auxin metabolites was performed using a high-performance liquid chromatography–electrospray tandem mass spectrometry with a 1260 Infinity II HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a reversed-phase column (Kinetex; 50 mm × 2.1 mm, 1.7 µm; Phenomenex) coupled to a 6495 Triple Quad detector (Agilent Technologies, Santa Clara, CA, USA). Individual analytes were detected in positive and negative ion mode using optimised conditions [56 (link)].
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