To analyze the JA content in TaWRKY13-A-OE and Col-0 Arabidopsis plants, fifth and sixth leaves of 4-week-old and 5-week-old Arabidopsis were selected for liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. A total amount of 200 mg leaves of the above plants were grounded and incubated with methanol for 24 h. By using the Oasis Max solid-phase extraction cartridge, all samples were purified. The JA content was measured using the ultra-performance liquid chromatography (UPLC) system (Waters) (Agilent Technologies Inc, California, USA) and QTRAP 6500 system (AB SCIEX, Framingham, MA, USA). The measurement of each sample was repeated in three biological replicates, and 2H5-JA was used as the internal reference. The JA content of each sample was finally examined by ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) (Waters) and QTRAP 6500 system (AB SCIEX).
Qtrap 6500 system
Manufactured by AB Sciex
Sourced in United States, Canada, Japan, China
The QTRAP 6500+ system is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument. It is designed to provide accurate and sensitive quantitative and qualitative analysis of a wide range of analytes. The system combines the functionality of a triple quadrupole mass spectrometer with the added capabilities of a linear ion trap, enabling enhanced selectivity and sensitivity for complex samples.
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Lab products found in correlation
Exionlc ad, by AB Sciex (6 mentions)
Analyst 1, by AB Sciex (6 mentions)
Acquity uplc beh c18 column, by Waters Corporation (3 mentions)
Nexera x2 uplc system, by Shimadzu (3 mentions)
Nexera x2 uhplc system, by Shimadzu (3 mentions)
Acquity uplc hss t3 c18 column, by Waters Corporation (2 mentions)
Nexera x2 uhplc, by Shimadzu (2 mentions)
6500 q trap, by Thermo Fisher Scientific (2 mentions)
Savant speedvac spd111v, by Thermo Fisher Scientific (2 mentions)
2 6 di tert butyl 4 methylphenol bht, by Merck Group (2 mentions)
Splash lipidomix mass spec standard, by Avanti Polar Lipids (2 mentions)
Beh c18 column, by Waters Corporation (2 mentions)
Uplc system, by Waters Corporation (2 mentions)
Hypersil gold column, by Thermo Fisher Scientific (1 mentions)
Multiquant software, by AB Sciex (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Hss t3 column, by Waters Corporation (1 mentions)
Glucose content assay kit, by Solarbio (1 mentions)
46 protocols using qtrap 6500 system
1
JA Content Analysis in Arabidopsis WRKY13 Overexpression
2
Lipidomic Analysis of Transduced CHO-K1 Cells
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CHO-K1 cells were harvested 72 h after transduction and lipidomic analyses of cell lysates were performed by shotgun lipidomics using the QTRAP6500+system (Sciex) as previously described34 (link).
3
Sensitive Amoxicillin Detection in Serum
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For the detection of amoxicillin, 200 μL of blood serum was spiked with 10 μL amoxicillin-d4 (50 μg/L serum) as internal standard, filled up to 1 mL with water/acetonitrile (90:10, vol/vol), vortexed, sonicated, and filtered using a 0.45 μm Phenex-RC membrane filter. The filtrates were directly analyzed using an Agilent 1260 Infinity LC coupled to a Sciex QTRAP-6500 system. Chromatographic separation was performed by injecting a 10 μL filtrate onto a Thermo Hypersil Gold column (150 × 2.1 mm, 3 μm). Within 10 minutes, the analysis was complete using the positive Electrospray ionization mode and the following transitions of multiple reaction monitoring: amoxicillin m/z 366 → 208 and m/z 366 → 349, amoxicilloic acid m/z 384 → 323 and m/z 384 → 367, amoxicillin diketopiperazine m/z 366 → 114 and m/z 366 → 160, amoxicillin-d4 m/z 370 → 212, and m/z 370 → 353. Method validation, which was carried out according to Commission Decision 2002/657/EC, showed linearity in the range of 2.5–80 μg/L, good reproducibility (<26%), and mean recoveries (103–116%) of 10 μg/L. The decision limits CCα, and the detection capabilities, were within the range of 5.3–6.2 and 7.8–11.7 μg/L.
4
Oxylipin Profiling from Serum Samples
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Serum samples from participants were immediately frozen at −80°C, then thawed once and immediately used for isolation of oxylipins. Aliquots of 100 μl were spiked with 200 μl of oxidized oxylipin extract, vortexed and the protein was precipitated. The solution was then spiked with 20 μl of 1 μM internal standard mixture. The oxylipins in the supernatants were extracted using Poly-Sery MAX SPE columns L (ANPE, Shanghai, China). Prior to analysis, the eluent was dried under vacuum and redissolved for UPLC-MS/MS analysis.
The sample extracts were analysed using a LC–electrospray inoization–MS/MS system (UPLC: ExionLC AD, Sciex, USA; MS: QTRAP 6500+ system, Sciex, USA). Linear ion trap and triple quadrupole scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP 6500+ LC-MS/MS system). Eicosanoids were analysed using scheduled multiple reaction monitoring. Data acquisition was performed using Analyst 1.6.3 software (Sciex), and multiquant software (Sciex) was used to quantify all oxylipins [22 (link), 23 (link)] (Supplementary Methods , available at Rheumatology online).
The sample extracts were analysed using a LC–electrospray inoization–MS/MS system (UPLC: ExionLC AD, Sciex, USA; MS: QTRAP 6500+ system, Sciex, USA). Linear ion trap and triple quadrupole scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP 6500+ LC-MS/MS system). Eicosanoids were analysed using scheduled multiple reaction monitoring. Data acquisition was performed using Analyst 1.6.3 software (Sciex), and multiquant software (Sciex) was used to quantify all oxylipins [22 (link), 23 (link)] (
5
Quantification of Drug Analytes by LC-MS/MS
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The concentrations of the extract solution from the filter plate and of the cell lysate were quantified using a tandem mass spectrometry QTRAP6500 System (SCIEX) coupled with an ACQUITY UPLC system (Waters) using the internal standard method. Mobile phases A and B used 10 mM of ammonium bicarbonate and acetonitrile, respectively. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm; Waters) at 50 °C, with the following gradient of mobile phase B: 1% (at 0.00 to 0.50 min), 1% to 95% (0.50 to 2.00 min), 95% (2.00 to 2.50 min) and 1% (2.51 to 3.00 min); the flow rate was 0.4 ml min−1. Mass spectrometric detection was performed by multiple reaction monitoring in the electrospray-ionization negative-ion mode controlled by Analyst 1.6.2, using m/z 443.1/364.9 for canagliflozin; 425.9/264.1 for TA-1887; 407.0/328.8 for dapagliflozin; 423.0/387.0 for sotagliflozin; 435.0/273.0 for phlorizin; 273.0/148.9 for phloretin; 192.9/100.9 for α-MG; and 439.0/309.1 for candesartan.
6
Metabolite Profiling of Plant Samples
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Extraction and measurements of sucrose, fructose, glucose and starch were performed according to Yang et al. [62 (link)].
Malate, citrate and isocitrate were extracted and determined according to Chen et al. [63 (link)].
Total soluble proteins were determined according to Bradford [64 (link)] after being extracted with 50 mM phosphate-buffer solution (pH 7.0).
Free amino acids and derivatives were assayed by Wuhan MetWare Biotechnology Co., Ltd. (Wu, China). Briefly, 50 mg of frozen sample was extracted with 500 μL of 70% (v/v) methanol/water (precooled at − 20 °C). The sample extracts were analyzed using an LC-ESI-MS/MS system (UPLC, ExionLC AD,https://sciex.com.cn /; MS, QTRAP® 6500+ System, https://sciex.com/ ).
Malate, citrate and isocitrate were extracted and determined according to Chen et al. [63 (link)].
Total soluble proteins were determined according to Bradford [64 (link)] after being extracted with 50 mM phosphate-buffer solution (pH 7.0).
Free amino acids and derivatives were assayed by Wuhan MetWare Biotechnology Co., Ltd. (Wu, China). Briefly, 50 mg of frozen sample was extracted with 500 μL of 70% (v/v) methanol/water (precooled at − 20 °C). The sample extracts were analyzed using an LC-ESI-MS/MS system (UPLC, ExionLC AD,
7
Targeted metabolomic analysis of blood samples
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The collected blood supernatants were sent to MetWare (Chengdu, China) for targeted metabolite analysis. After thawing the blood sample on ice, 300 μl of pure methanol was added to 50 μl of sample to remove blood protein. The samples were then centrifuged at 12000 g and 4°C several times (centrifuged 10 min → collected the supernatant → centrifuged 5 min → stored at 20° for 30 min → centrifuged 3 min). In total, 150 μl of supernatant was analyzed using high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS; UPLC, ExionLC AD; MS, QTRAP 6500 + System, Sciex). All chemicals used, including methanol (Merck), acetonitrile (Merck), formic acid (Aladdin), and standards (BioBioPha/Sigma-Aldrich), were chromatographically pure. Analyst v1.6.3 was used to analyze mass spectrometry data, in a qualitative analysis of the metabolites according to the retention time of the detected substance and secondary spectral data based on the MetWare database.
Each metabolite was subjected to a two-tailed unpaired t-test assuming unequal variances and using the q-value package (for total metabolites) to correct for false discovery.
Each metabolite was subjected to a two-tailed unpaired t-test assuming unequal variances and using the q-value package (for total metabolites) to correct for false discovery.
8
Quantitative Metabolite Profiling of Leaf Tissue
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Leaf tissue (50 mg) was ground in 1.0 ml of 50% acetonitrile (plus internal standard) in a grinding tube with magnetic beads (50 Hz, 5 min), followed by rotary extraction (1 h, 4°C). After centrifugation (12000g, 15 min, 4°C), the supernatant was filtrated, freeze-dried and redissolved in 50 μl of 50% acetonitrile. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed using a QTRAP 6500+ system (SCIEX) at the BGI (Shenzhen, China). Samples (10 μl) were injected into the HSS T3 Column (Waters, USA) and eluted using eluent A (0.02% formic acid) and eluent B (0.02% formic acid + acetonitrile). The solvent gradient was set as 0–3 min 2% B, 3–15 min 80% B, 15–18 min 98% B, 18–20 min 2% B. Ion source parameters were 500 °C, 5000 V, curtain gas 30 psi, gas I 40 psi, gas II 40 psi, collision gas 8 psi for positive mode, and 450°C, –4500 V, 35, 55, 45, and 9 psi for the negative mode. Multiple reaction monitoring was performed for metabolite identification and quantification using the Novogene database.
9
Metabolomic Profiling of CD31+ Cells
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To extract metabolites and remove proteins, ECs isolated from tissues were first washed with precooled PBS twice and then resuspended in a 500 µL mixture of the same volume of cold methanol and acetonitrile (Merck, GER). Next, the cell suspension was centrifuged at 14 000 × g and 4 °C for 20 min, and then 300 µL supernatant was transferred into a new centrifuge tube, which was incubated at −20 °C for 30 min. Then, the supernatant was recentrifuged at 14 000 × g and 4 °C for 10 min, and 200 µL of supernatant was transferred through a protein precipitation plate for further LC–MS analysis. Targeted metabolomics profiling of CD31+ cells and targeted metabolite detection were performed using an LC–ESI–MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/; MS, QTRAP 6500+ System, https://sciex.com/ ).
The concentrations of amino acids and glucose in BCa cells and tissues were, respectively, determined by a micro amino acid content assay kit (Solarbio, China) and a glucose content assay kit (Solarbio, China) according to the manufacturer's instructions.
The concentrations of amino acids and glucose in BCa cells and tissues were, respectively, determined by a micro amino acid content assay kit (Solarbio, China) and a glucose content assay kit (Solarbio, China) according to the manufacturer's instructions.
10
Amide Column LC-ESI-MS/MS Analysis
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An LC-ESI-MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/ ; MS, QTRAP® 6500 + System, https://sciex.com/ ) was used to analyze the sample extracts. The HPLC column used was ACQUITY BEH Amide (i.d.2.1 × 100 mm, 1.7 μm) and the solvent system consisted of water with 2 mM ammonium acetate and 0.04% formic acid (A) and acetonitrile with 2 mM ammonium acetate and 0.04% formic acid (B). The gradient was initiated at 90% B (0-1.2 min), decreased to 60% B (9 min), 40% B (10–11 min) and finally ramped back to 90% B (11.01-15 min). The flow rate was set at 0.4 mL/min and the temperature was maintained at 40 °C. The injection volume used was 2 µL.
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