Pegasus ht time of flight mass spectrometer

Frozen leaves of wheat seedlings from each biological replicate were homogenized using a mortar and pestle with liquid nitrogen. The metabolites of the leaves (60 ± 1 mg of fresh weight) were extracted with 0.48 mL of 75% methanol, then 24 μL of adonitol was added to the sample as internal standard. Secondly, the samples were homogenized and centrifuged for 15 min at 13000 rpm. The supernatants from each sample were taken and dried in a vacuum concentrator without heating and then dissolved with 60 μL of methoxy amination hydrochloride (20 mg/mL in pyridine) to be incubated for 30 min at 80°C. Then, the sample aliquots were further incubated with 80 μL of the N-methyl-N-(trimethylsilyl)trifluoroacetamide (BSTFA) for 2 h at 70°C. Finally, the mixture was used for GC-TOF-MS analysis (Dunn et al., 2011 (link)). The separation and quantification of organic acids, carbohydrates, and amino acids were performed by Agilent 7890 gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer according to Wu et al. (2013) (link). Nitrate was quantified by the method described by Dutilleul et al. (2005) (link). The contents of ammonium were measured by the phenol hypochlorite assay (Tercé-Laforgue et al., 2004 (link)).

Gas Chromatography Tandem Time-of-Flight Mass Spectrometry (GC-TOF-MS) is the basis of metabonomic data analysis [16 (link), 17 (link)]. GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer. The system utilized a DB-5MS capillary column coated with 5% diphenyl cross-linked with 95% dimethylpolysiloxane (30 m×250 μm inner diameter, 0.25 μm film thickness; J&W Scientific, Folsom, CA, USA). 1 μL aliquot of the analyte was injected in splitless mode. Helium was used as the carrier gas, the front inlet purge flow was 3 mL/min, and the gas flow rate through the column was 1 mL/min. The initial temperature was kept at 50°C for 1min, then raised to 310°C at a rate of 10°C/min, and then kept for 8min at 310°C. The injection, transfer line, and ion source temperatures were 280, 270, and 220°C, respectively. The energy was -70 eV in electron impact mode. The mass spectrometry data were acquired in full-scan mode with the m/z range of 50-500 at a rate of 20 spectra per second after a solvent delay of 6.04 min.

A volume of 0.48 mL methanol–water (3:1, v/v) and 24 μL of adonitol (1 mg/mL stock in dH₂O) were added to 0.06 g of each sample in a 2 mL Eppendorf tube as internal standard, followed by homogenization in a ball mill for 4 min at 50 Hz, and then sonication for 5 min twice (with incubation in ice water). After centrifugation at 13000×g at 4 °C for 15 min, 350 μL of supernatant was transferred into a fresh 2 mL GC/MS glass vial. After drying the samples with a vacuum concentrator, 80 μL methoxyamine hydrochloride (20 mg/mL in pyridine) was added to each sample and incubated at 80 °C for 30 min. Then, 100 μL of the BSTFA regent (1% TMCS, v/v) was added and the mixture incubated at 70 °C for 1.5 h and mixed well for GC–time-of-flight (TOF)–MS analysis.
GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Each treatment in this metabolomics study was repeated with at least six biological replicates.
Chroma TOF 4.3X software of LECO Corporation and LECO-Fiehn Rtx5 database were used for raw peaks exacting, data baselines filtering, baseline calibration, peak alignment, deconvolution analysis, peak identification and integration of the peak area. The RI (retention time index) method was used for peak identification, with the RI tolerance at 5000.

The serum sample preparation procedure and GC-TOF-MS analysis were similar to the previously published procedure with slight modification (Gu et al., 2015 (link); Geng et al., 2020 (link); Yu et al., 2020 (link)). In brief, take 50 μl of sample and mix it with 5 μl of internal standard (L-2-chlorophenylalanine) into 1.5-ml Eppendorf (EP) tubes, and 200 μl of methanol was added. Treat with ultrasound for 10 min (incubated in ice water). Centrifuge for 15 min at 12,000 rpm and 4°C. Transfer the supernatant of 180 μl into fresh 1.5-ml EP tubes. Take 20 μl from each sample and pool as QC sample. The dried samples (in a vacuum concentrator) were then incubated with 30 μl of methoxy amination hydrochloride at 80°C for 30 min. Subsequently, 40 μl of the bis (trimethylsilyl)trifluoroacetamide (BSTFA) reagent was added to the sample aliquots and incubated for 1.5 h at 70°C. Then, 5 μl of fatty acid methyl esters (FAMEs) (in chloroform) was added to the QC sample at room temperature. Finally, an Agilent 7890 gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer was used to perform GC-TOF-MS analysis. The energy was −70 eV in electron impact mode. After 4.7 min of solvent delay, mass spectrometry data were obtained at full-scan mode (m/z 50–500) at a rate of 12.5 spectra per second.

An Agilent 7890 gas chromatograph system equipped with a Pegasus HT time-of-flight mass spectrometer was used to perform GC-TOF-MS analysis. The 1 μl analyte was analyzed using the splitless mode. The carrier gas was Helium, and 3 ml min⁻¹ was set as the front inlet purge flow, and 1 ml min⁻¹ was set as the gas flow rate through the column. The initial temperature was maintained at 50 °C for 1 min, followed by increase at a rate of 10 °C min⁻¹, until reaching 310 °C, when it was maintained for 8 min. Injection temperature was 280 °C, the transfer line temperature was 270 °C, and the ion sources temperature was 220 °C. The energy of electron impact mode was −70 eV. Mass spectrometry data were generated in full-scan mode using an m/z range of 50–500, with 20 spectra per second following a 366-s solvent delay.

A global metabolomics analysis was conducted using gas chromatography-mass spectrometry (GC – MS) and ultra-performance liquid chromatography – MS (UPLC – MS). The GC – MS analysis was performed using an Agilent 7890B gas chromatograph equipped with PEGASUS HT time-of-flight mass spectrometer (LECO). UPLC – MS analysis was conducted using an Agilent 1290 Infinity UPLC and AB triple TOF 5600\/6600 mass spectrometer (AB SCIEX). Sample equivalents were mixed to prepare quality controls (QCs) for the assessment of reproducibility. Detailed sample preparations, GC – MS and UPLC – MS analyses, and QC preparation are described in the Methods section of Supplementary material. Ion peaks generated from GC – MS were annotated with LECO-Fiehn Rtx5 database, whereas those from UPLC – MS were annotated with the MetDDA database and LipDDA methods. All data were log-transformed for normalization before statistical analysis.

GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph system equipped with a Pegasus HT time-of-flight mass spectrometer. The system has a DB-5MS capillary column coated with 5% diphenyl cross-linked with 95% dimethylpolysiloxane (30 m × 250 μm inner diameter, 0.25 μm film thickness; J&W Scientific, Folsom, CA, USA). A 1 μL aliquot of the analyte was injected in the splitless mode. Helium was used as the carrier gas with a purge flow rate of 3 mL min⁻¹ at the front inlet and a gas flow rate of 1 mL min⁻¹ through the column. The initial temperature was kept at 50°C for 1 min, then raised to 310°C at a rate of 20°C min⁻¹, then kept for 6 min at 310°C. The temperature of injection, transfer line, and ion source were 280, 280, and 250°C, respectively. The energy in the electron impact mode was -70 eV. The mass spectrometry data were obtained in the full-scan mode with the m/z range of 50-500 at a rate of 12.5 spectra per second after a solvent delay of 4.83 min.