Mass spectrometry data were acquired on an Agilent 6520 Accurate-Mass Q-TOF LC/MS System (Agilent Technologies, Inc.) equipped with a dual electro-spray source, operated in the negative-ion mode. Separation was performed on Clarity 2.6u Oligo-MS 100 A column (2.1 mm × 100 mm). Mobile phase buffer A consisted of 45 mM hexafluoroisopropanol (HFIP), 3 mM triethylamine (TEA) and 10 µM EDTA in water at pH 7.3. Mobile phase buffer B consisted of 45 mM HFIP, 3 mM TEA and 10 µM EDTA in 90% methanol/water. The analytes were eluted at a flow rate of 0.2 ml min−1 with a 10 to 50% organic gradient exchange over 15 min. The instrument was used in full-scan TOF mode. MS source parameters were set with a capillary voltage of 4 kV, the fragmentor voltage of 200 V and skimmer 65 V. The gas temperature was 350 °C, drying gas flow 10 l min−1 and nebulizer pressure 25 psig. Data were acquired at high resolution (3,200 m/z), 4 GHz. TOF-MS mass spectra were recorded across the range 200–3,200 m/z. Data acquisition and analysis were performed using MassHunter Workstation Software (version B.07.00). To maintain mass accuracy during the run time, an internal mass calibration sample was infused continuously during the liquid chromatography–mass spectrometry (LC/MS) runs.
Agilent 6520 accurate mass q tof lc ms system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6520 Accurate-Mass Q-TOF LC/MS System is a high-performance liquid chromatography-mass spectrometry (LC-MS) instrument. It provides precise mass measurements and high-resolution identification of molecular compounds.
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5 protocols using agilent 6520 accurate mass q tof lc ms system
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High-Resolution Q-TOF LC-MS Protocol
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PvAMA1 Protein Characterization by LC-MS/MS
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Recombinant PvAMA1 ectodomain was electrophoresed using 12% SDS-PAGE under non-reduced conditions followed by staining with Coomassie Blue. The band of interest was excited from the gel and reduced (20 mM Tributylphosphine), alkylated (40 mM Iodoacetamine), and in-gel trypsin digestion, and then sent for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis (Monash University Malaysia) for protein identification. The LC-MS/MS was performed using Agilent 6520 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA). Peptide ions were analyzed with GPS Explorer software (Applied Biosystems, Foster City, CA, USA) using MASCOT™ Database.
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Metabolite Identification in Fungal Extracts
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To identify the metabolites in the fungal extracts, a mass spectrometry was performed on an Agilent 6520 Accurate Mass Q-TOf LC/MS system (Agilent, Santa Clara, CA, USA) equipped with an electrospray ionization interface, and operated in positive ion mode with parameters set as follows: capillary voltage, 3500 V; fragmenter, 100 V; pressure of nebulizer, 35 psig; drying gas temperature, 300 °C; acquisition range 150–800 m/z. Nitrogen was used as the drying gas at a flow rate of 12.0 L min−1. The system was also equipped with a diode array detector operating from 280 to 350 nm with a step of 2 nm. Samples were eluted on an Agilent Zorbax eEclipse Plus C18 Rapid Resolution column (4.6 × 100 mm 3.5 μm). The column temperature was maintained at 30 °C. A mobile phase consisting of 0.1% formic acid in ultrapure water (obtained from Millipore Integral-5 purification system) (solvent A) and 0.1% formic acid in acetonitrile (solvent B) was applied with the following gradient: 0–10% B (0 min), 10–100% B (30 min), 100% B isocratic mode (10 min) and, for column re-conditioning 100–10% B (1 min) and 10% B (7 min). Both formic acid and acetonitrile were of LC/MS grade. The flow rate was set at 0.30 mL min–1 and the injection volume was 1 μL.
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Purification and Characterization of Organic Compounds
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The reagents were purchased from commercial chemical reagent companies and used without further purification. Column chromatography was carried out on silica gel (200–300 mesh). 1H NMR and 13C NMR spectra were recorded on a 400 MHz spectrometer (Bruker Magnet System 400’ 54 Ascend). Melting points were measured on a BUCHI B-540 and uncorrected. HRMS (ESI) was recorded using Agilent 6520 accurate-Mass Q-TOF LC/MS system (1200–6520/Agilent).
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Accurate-Mass Q-TOF LC/MS Metabolomics
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Mass spectrometry data were acquired on an Agilent 6520 Accurate-Mass Q-TOF LC/MS System, (Agilent Technologies, Inc., Santa Clara, CA) equipped with a dual electro-spray source, operated in the positive-ion mode. Separation was performed on Zorbax 300SB-C3 Poroshell column (2.1 mm x 75 mm; particle size 5 μm). The analytes were eluted at a flow rate of 1 ml/min with a 5 to 90% organic gradient over 5 minutes and holding organic for 1 minute. Both mobile phases, water and acetonitrile, contained 0.1% formic acid. The instrument was used in a full-scan TOF mode. MS source parameters were set with a capillary voltage of 4 kV, the fragmentor voltage of 220 V and skimmer 65 V. The gas temperature was 350 °C, drying gas flow 12 l/min and nebulizer pressure 55 psig. Data were acquired at high resolution (3,200 m/z), 4 GHz. TOF-MS mass spectra were recorded across the range 100–3,200 m/z. To maintain mass accuracy during the run time, an internal mass calibration sample was infused continuously during the LC/MS runs. Data acquisition was performed using Mass Hunter Workstation (version B.06.01). For data analysis and deconvolution of mass spectra Mass Hunter Qualitative Analysis software (version B.07.00) with Bioconfirm Workflow was used.
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