L-Erg and S-Met-L-Erg determination in the urines of the test experiment was carried out as previously described in Ref. [26 (link)]. The analysis of L-Erg and S-Met-L-Erg in the urines of low and high-dose treatment was performed on an Agilent 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, USA) coupled to a 6500 QTRAP mass spectrometer equipped with Ion Drive Turbo V ion source LC-MS/MS system (Sciex, Framingham, MA, USA). L-Erg and S-Met-L-Erg concentration was normalized by urinary creatinine, determined with the Creatinine Assay Kit (Sigma-Aldrich, MA, USA).
Agilent 1290 infinity 2 uhplc system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1290 Infinity II UHPLC system is a high-performance liquid chromatography (HPLC) instrument designed for efficient and precise separation of complex samples. It features advanced technology for improved resolution, sensitivity, and throughput. The system is capable of operating at ultra-high pressures, enabling the use of smaller particle size columns for enhanced separation performance.
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Lab products found in correlation
6470a triple quadrupole mass spectrometer, by Agilent Technologies (2 mentions)
Creatinine assay kit, by Merck Group (1 mentions)
Ion drive turbo 5, by AB Sciex (1 mentions)
6550 ifunnel q tof lc ms system, by Agilent Technologies (1 mentions)
C18 column, by Agilent Technologies (1 mentions)
Orbitrap eclipse tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer 2.4 sp1, by Thermo Fisher Scientific (1 mentions)
6545 quadrupole time of flight q tof mass spectrometer, by Agilent Technologies (1 mentions)
Masshunter b 06, by Agilent Technologies (1 mentions)
Acquity uhplc beh c18 column, by Waters Corporation (1 mentions)
15 t solarix xr ft icr mass spectrometer, by Bruker (1 mentions)
Xselect hss t3, by Waters Corporation (1 mentions)
Simca p 14, by Sartorius (1 mentions)
Agilent masshunter qualitative analysis b 08, by Agilent Technologies (1 mentions)
Spermine, by Merck Group (1 mentions)
Spermidine, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
L ornithine, by Merck Group (1 mentions)
L arginine, by Merck Group (1 mentions)
Acquity uplc beh c18 column, by Agilent Technologies (1 mentions)
22 protocols using agilent 1290 infinity 2 uhplc system
1
Quantification of L-Erg and S-Met-L-Erg in Urine
2
Bioactive Compound Analysis in P. speciosa
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The analyses of bioactive compounds present in P. speciosa extracts were performed using Agilent 1290 Infinity II UHPLC System (Santa Clara, CA, USA) and 6550 iFunnel Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA). Separation was carried out using a C18 column (100 mm × 2.1 mm I.D.; 3 μm particle size) (Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of 0.1% formic acid in water (solvent A) and methanol (solvent B) at a flow rate of 0.4 mL/min at 25 °C. The elution conditions were: 0 min, 85% A; 2–10 min, 50% A; 15 min, 30% A; 20 min, 15% A; 22–23 min, 0% A; 26 min, 85% A. MS detection was carried out in negative ion mode using a mass range of 100–1200 m/z. The negative ion mode was chosen because it appeared more selective and more sensitive for further LC-MS analysis of flavonoids and phenolics in plants. The extracted samples were dissolved in methanol, thoroughly mixed, filtered with a nylon filter of 0.45 μm and analyzed.
3
HDX Analysis of CFTR Conformational Changes
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Before the HDX experiments, P67L-CFTR-6SS was pre-incubated at 25 °C for 5 min in the absence or presence of 30 µM VX-809 or VX-445 correctors. DMSO (0.1%) was included in all conditions. HDX was initiated by mixing pre-incubated P67-CFTR-6SS into D2O-based buffer pre-incubated at 37 °C, at 1:9 dilution. The corrector concentration was kept at 30 µM during HDX incubation for 10 and 240 s at 37 °C. The HDX reaction was quenched by adding a 10 μl aliquot of the mixture into 5 μl of chilled quenching buffer (1 M glycine-HCl including 0.02% DDM, pH 2.4). Quenched samples were stored at −80 °C. The on-line pepsin digestion was carried at 60 μl/min flow rate for 1.5 min at 15 °C, and desalting was performed at a 200 μl/min flow rate for 1.5 min. Digestion mixtures were separated by an Agilent 1290 Infinity II UHPLC system using a 5–40% liner gradient of ACN containing 0.1% FA for 11 min at 65 μl/min. MS measurements were performed using an Orbitrap Eclipse tribrid mass spectrometer (ThermoFisher Scientific). Mass spectra of peptides were acquired in positive-ion mode for m/z 200–2000. Data analysis was carried out using HDExaminer 3.3 (Sierra Analytics). MS/MS spectra were analyzed using Proteome Discoverer 2.4 SP1 (ThermoFisher Scientific). All HDX data collected are included in Source Data file.
4
Quantitative UHPLC-QTOF MS Analysis
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An Agilent 1290 Infinity II UHPLC system (Agilent Technologies, Palo Alto, CA, USA), coupled with a Waters Acquity HSS T3 (2.1 × 100 mm × 1.8 um) separation column (Waters, Milford, MA, USA) and a HSS T3 (2.1 × 5 mm × 1.8 um) guard column, was used to obtain the chromatographic separations with the column temperature set at 40 °C. The sample injection volume was 5 μL. The mobile phases consisted of solvents A (0.1% formic acid in ddH2O) and B (0.1% formic acid in acetonitrile) with a 0.45 mL/min flow rate. The gradient program was as follows: 0–1 min, 100% A; 1–16 min, a linear gradient to 70% A; 16–21 min, a linear gradient to 5% A, and hold for 1.5 min; returning to 100% A over 1 min, and hold for 5 min for column re-equilibration. An Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer was used for mass spectrometric analysis. The positive electrospray ionization (ESI) mode was applied for mass spectral (70–1000 m/z) data collection. The ESI capillary voltage was 3.5 kV, the drying gas flow rate was 8.0 L/min, the temperature of the nitrogen gas was 325 °C, the nebulizer gas pressure was 30 psig, the fragmentor voltage was 130 V, the skimmer was 45 V, and the Oct 1 RF Vpp was 750 V. Mass data were acquired using Agilent MassHunter B.06.
5
Metabolite Profiling Using UHPLC-QTOF
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Structural characterisation of metabolites and metabolite profiling was performed using an Agilent 1290 Infinity II UHPLC system connected to an Agilent 6560A IM QTOF system (Agilent Technologies, Santa Clara CA, USA) fitted with an electrospray ionisation (ESI) source operated in positive-ion mode over a mass range m/z 50–1000. Reversed-phase gradient elution was performed on an Acquity U(H)PLC BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters) at room temperature. The mobile phases consisted of 0.1% FA in water (A) and ACN (B). The gradient used was as follows: 10% B at 0 min increased to 70% B at 6.0 min. The solvent composition was held at 70% B for 0.5 min and then decreased to 10% B at 6.7 min. For analysis, 10 µL of each sample was injected. Full-scan MS spectra were obtained over the mass range 50–1000 Da. Targeted MSMS spectra were acquired on selected metabolites at medium isolation width (~ 4 m/z) using a collision energy of 17 V.
6
High-Resolution Mass Spectrometry Analysis
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The supernatants of the reaction mixture were injected and subsequently desalted on-line using reverse-phase microtrap column (Luna Omega 5um Polar C18, 100 Å, 20 × 0.3 mm, Phenomenex). The analytes eluted from the microtrap were separated on the reverse-phase analytical column (Luna Omega 3um Polar C18, 100 Å, 150 × 0.3 mm, Phenomenex) at 50 °C using Agilent 1290 Infinity II UHPLC system at flow rate 10 µl/min in water/acetonitrile gradient (mobile phase [A] was 98% water and 2% acetonitrile with 0.1% formic acid; mobile phase [B] was 98% acetonitrile and 2% water with 0.1% formic acid; the gradient started at 5% [B] and reached 90% [B] in 30 min). The eluted analytes were analyzed by 15 T solariX XR FT-ICR mass spectrometer (Bruker Daltonics). Mass spectral data were collected in positive broadband mode over the m/z range 150–1500, with 1 M data points transient and 0.2 s ion accumulation with two averaged scans per spectrum. Data acquisition and data processing were performed using ftmsControl 2.1.0 and DataAnalysis 5.0.
7
Metabolomic Profiling of Flower Development
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Petals at stage 1 and stage 4 of V1, V2, and V3 were prepared, named V1-1, V1-4, V2-1, V2-4, V3-1, and V3-4, respectively, and 3 biological replicates were set. About 50 mg sample was weighed out for supernatant preparation. Agilent 1290 Infinity II UHPLC system coupled to an Agilent 6545 UHD and Accurate-Mass Q-TOF/MS was used for liquid chromatography-mass spectrometry (LC-MS) analysis. The chromatographic column used was Waters XSelect HSS T3 (2.5 μm, 100 mm × 2.1 mm). Raw data were converted to common (mz.data) using Agilent MassHunter Qualitative Analysis B.08.00 software (Agilent Technologies, USA). Then all data went through internal standard normalization and weight normalization. Visualization matrices containing sample name, m/z-RT pair, and peak area were obtained. After editing, the data matrices were imported into SIMCA-P 14.1 (Umetrics, Umea, Sweden), mean-centered and scaled to Pareto variance. Then, a multivariate analysis was conducted. |log2FoldChange|>1 and p ≤ 0.05 were determined as differentially accumulated metabolites (DAMs). DAMs Venn diagram, principal component analysis (PCA), Gene Ontology (GO), and KEGG enrichment (Kyoto Encyclopedia of Genes and Genomes, KEGG) were also conducted for further analysis.
8
Quantitative Polyamine Analysis in ESCC Cells
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The ESCC cells with/without Fn infection for 24 h were collected in 80% aqueous methanol. After ultrasonic treatment, the lysates were centrifuged, and the supernatant was dried and dissolved in 50 μL of 100 mM sodium carbonate. The chemical derivatization was initiated after adding 50 μL of 2% benzoyl chloride in acetonitrile. After derivatization, the sample was isometrically mixed with benzoyl-13C6 chloride-derivatized standards (as internal standards) prior to ultra-high-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) analysis. The UHPLC-MS/MS analysis was performed on an Agilent 1290 Infinity II UHPLC system coupled to a 6470A Triple Quadrupole mass spectrometer (Agilent, Santa Clara, United States).
The reagents used to prepare for standard solution, such asl- arginine, l- ornithine, putrescine, spermine, and spermidine, were purchased from Sigma-Aldrich. N1-acetylspermidine and N1-acetylspermine were purchased from Shanghai Yuanye Bio-Technology Co (Shanghai, China).
The reagents used to prepare for standard solution, such as
9
Quantitative HPLC Analysis of Resveratrol
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The LC separation was performed using hydrophilic interaction chromatography with an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm, Agilent Technologies) operated by Agilent 1290 Infinity II UHPLC system (Agilent, Santa Clara, USA). The mobile phase consisted of A (water containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid) and was run in gradient elution i.e., 0–4 min, 90% B; 4–11 min, 90% B and 9–15 min, 10% B. The flow rate and injection volumes were 0.25 ml/min and 2 µl, respectively. Elution was monitored at 280 nm by using a PDA detector and column temperature was maintained at 30 ®C. Detection of resveratrol and trans-stilbene was performed by spectral matches with standards of resveratrol and trans-stilbene (Sigma-Aldrich) prepared in methanol (1.0 mg/ ml; w/v).
10
UHPLC-DAD Analysis of Medicinal Materials
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UAE was performed with a KQ5200E ultrasonic cleaner (Kunshan Ultrasound Instrument Co., Ltd., Kunshan, China). UHPLC-DAD analysis of medicinal materials and preparations was performed on an Agilent 1290 infinity II UHPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a 1290 DAD detector and a 1290 vial sampler using Agilent ZORBAX Eclise Plus C18 column (2.1 mm × 100 mm, 1.8 μm) coupled with an Agilent UPLC guard 3PK (2.1 × 5 mm, 1.8 μm). The mobile phases for UHPLC-DAD analysis were water containing 0.1% formic acid (A) and acetonitrile (B), and the gradient elution was 15% B (0–3 min), 15–18.5% B (3–4 min), 18.5% B (4–8 min), 18.5–24% B (8–10 min), 24% B (10–16 min), 24–30% B (16–17 min), 30% B (17–19 min), 30–38% B (19–20 min), 38–40% B (20–23 min), 40% B (23–25 min), 40–44% B (25–26 min) and 44% B (26–28 min) with a flow rate of 0.3 mL/min, and the column temperature was controlled at 30 °C. Meanwhile, the online monitoring wavelengths were 0–7.3 min (348 nm), 7.3–9.0 min (284 nm), 9.0–13.0 min (330 nm), 13.0–23.3 min (270 nm), 23.3–24.7 min (250 nm) and 24.7–28 min (280 nm). The injection volume was 3 μL.
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