Quanlynx software

Crude bioreactor media was centrifuged and passed through a 0.22 μm filter. A perchloric acid cleanup was used to remove protein and particulate matter, which involved mixing filtered bioreactor media with 0.4 N HClO₄ at a 1:1 ratio and centrifuging at 1962g for 5 min at RT. The clarified media was collected to be analyzed by LC‐MS.
A Waters Xevo G2 Q‐ToF (run in ESI‐positive sensitivity mode) coupled to a Waters ACQUITY UPLC I‐Class was used for analysis. We used an Intrada Amino Acid column (Imtakt) to perform normal phase chromatography and separate the amino acids. The buffers used were A: acetonitrile + 0.1% formic acid and B: 100 mM ammonium formate, with a flow rate of 0.6 ml/min, a gradient time of 15 min, and column temperature of 40°C. Amino Acid Standards (Agilent) were utilized to generate a calibration curve 20–2700 pmol/μL) in the QuanLynx software (Waters), which was used to calculate the concentrations of amino acids detected in the prepared bioreactor media samples. Media samples were run in triplicate, with error bars indicating standard deviations. Additional information on this method can be found in past work (Velugula‐Yellela, Kohnhorst, et al., 2018).

Data were acquired and processed using MassLynx 4.1 and QuanLynx software (Waters, Milford, MA, USA). The DA concentrations in samples (µg·g⁻¹) were calculated directly from the area responses using a linear seven-point calibration ranged from 0.005–4 µg·mL⁻¹. The confirmatory guidelines for DA were in agreement with the performance criteria of European Commission (EC) Decision 2002/657/EC [39 ]. The maximum permitted tolerances for the relative retention time and relative ion intensities (ion ratio) should be within ± 2.5% and ± 25%, respectively.

Determination of plasma lactulose and d-mannitol concentrations were performed in an Acquity UPLC (Waters Corp., Mildford, MA, USA) connected to a Xevo-G2 Qtof mass spectrometer (Waters Corp., Mildford, MA, USA) operating in full scan negative mode (100 to 1200 m/z). Chromatographic separation was achieved with a linear gradient using a BEH amide column (21 × 100 mm, 1.7 µm, Waters Corp., Mildford, MA, USA) and mobile phases comprising A = 10 mM ammonium acetate in ACN:H₂O (9:1) and B = 10 mM ammonium acetate in ACN:H₂O (4:6). Flow rate was set to 0.5 ml/min and oven temperature to 40 °C. Leucine-enkephalin (200 ng/ml) was used as lock mass. Data processing was performed with QuanLynx software (Waters Corp., Mildford, MA, USA). Ion areas were used for quantification based on standard curves prepared using authentic standards and raffinose as internal standards (IS). Plasma samples (25 µL) were submitted to protein precipitation with 56 µL of IS solution. Four independent replicates per sample were prepared and analyzed.

Metabolome analysis was performed as previously described [58 (link)]. Four pools of 20 seedlings per condition were used for metabolome analysis. Approximately 30 mg of the ground frozen seedling samples was analyzed by an Agilent 7890A gas chromatograph (GC) coupled to an Agilent 5975C mass spectrometer (MS). Standards were injected at the beginning and end of the analysis. Data were analyzed with AMDIS (http://chemdata.nist.gov/mass-spc/amdis/ accessed on 5 December 2013) and QuanLynx software (Waters Corp., Milford, MA, USA).

JA and JA-IL levels were quantified using an ethyl acetate extraction method in conjunction with HPLC/MS similar to that described in Chung et al. (2008 (link)). Briefly, samples (approximately 150 mg tissue) were frozen in liquid nitrogen and hormones were extracted using 1 mL of extraction solvent (80:20 methanol:water + 0.1% formic acid) for 18 h at −20 C. Extracts were then centrifuged (10,000 × g for 10 min at 4°C) and the supernatant was transferred to autosampler vials. Five μL of each supernatant were injected into a Waters UPLC BEH C18 column (2.1 × 50 mm; 1.7 μm particles) held at 50°C on a Waters (Milford, MA, USA) Acquity ultraperformance liquid chromatography (UPLC) system that was coupled to a Waters Quattro Premier XE tandem quadrupole mass spectrometer. Separation was performed using a linear gradient based upon 0.15% aqueous formic acid (A) and methanol (B) over a 3-min program using a total flow rate of 0.4 mL/min. Quantification of JA and SA was performed using electrospray ionization in negative-ion mode using multiple reaction monitoring (MRM), using m/z 209 ≥ 59 for JA, m/z 322 ≥ 130 for JA-IL (Chung et al., 2008 (link)), and m/z 137 ≥ 93 for SA (Zeng et al., 2011 (link)). Peak areas were integrated, and calibration curves generated, using Waters QuanLynx software.

For amino acid analysis by LC–MS, crude bioreactor media was centrifuged and passed through a 0.22 μm filter. A perchloric acid cleanup was used to remove protein and particulate matter, which involved mixing filtered bioreactor media with 0.4 N HClO₄ at a 1:1 ratio and centrifuging at 1,962g for 5 min at RT.20 The clarified media was collected to be analyzed by LC–MS.
A Waters Xevo G2 Q‐ToF (run in ESI positive sensitivity mode) coupled to a Waters ACQUITY UPLC I‐Class was used for analysis. We used an Intrada Amino Acid column (Imtakt USA) (100 × 2 mm, 3 μm particles) to perform normal phase chromatography and separate the amino acids. The buffers used were A: acetonitrile + 0.1% formic acid and B: 100 mM ammonium formate, with a flow rate of 0.6 ml/min, a gradient time of 15 min, and column temperature of 40°C. Amino Acid Standards (Agilent) were utilized to generate a calibration curve (9 to 900 pmol/ l) in the QuanLynx software (Waters), which was used to calculate the concentrations of amino acids detected in the prepared bioreactor media samples. Media samples were run in triplicate, with error bars indicating SDs. Injection order was randomized to eliminate order bias. Additional information on this method can be found in past work.20