Cells were grown in 10 cm2 dishes. Following 24 h of treatment with DMOG or vehicle, media was aspirated, and cells were quickly rinsed with ice cold normal saline. Culture dishes were placed on dry ice and 600μL ice cold extraction buffer (40:40:20 acetonitrile/methanol/200 mM NaCl, 10 mM Tris-HCl, pH 9.2) were added directly to the cells. Cells were scraped and transferred to 1.5 mL tubes and were frozen in liquid nitrogen. Cells were subjected to two freeze thaw cycles with vortex of 15 s after each thaw. After lysis, cell lysates were centrifuged at 20,000xg for 15 min at 4°C. Metabolite containing supernatant was transferred to fresh 1.5 mL tube and protein pellets were used for protein quantification. The metabolite containing supernatants were dried down by a SpeedVac concentrator (Thermo Savant). After reconstitution in 60% acetonitrile, samples were analyzed by Thermo Fisher Scientific U3000 UHPLC equipped with Q ExactiveTM Q-Orbitrap MS.
Q exactive q orbitrap ms
Manufactured by Thermo Fisher Scientific
The Q Exactive Q-Orbitrap MS is a high-resolution, accurate-mass (HRAM) mass spectrometer. It combines a quadrupole mass filter with an Orbitrap mass analyzer to provide high-resolution, accurate mass measurements of analytes.
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Lab products found in correlation
2 protocols using q exactive q orbitrap ms
1
Metabolite Extraction and Mass Spectrometry Analysis
2
Untargeted Metabolomics with UHPLC-Q Exactive
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Untargeted metabolomics was accomplished with Thermo Fisher Scientific U3000 UHPLC equipped with Q ExactiveTM Q-Orbitrap MS through an HESI source. Chromatographic separation was achieved on a Waters BEH C18 column (2.1 × 100 mm, 1.7 μm). Temperature for the column oven and automatic sampler was set at 35 and 4°C, respectively. The mobile phase consisted of 0.1% acetic acid in water (A) and acetonitrile (B) with a flow rate of 0.4 mL ⋅ min-1, running in accordance with a gradient ranging from 3 to 97% of B within 20 min. The injection volume was 2 μL.
High-accuracy MS data were acquired in the negative mode by Full MS/dd-MS2. The HESI source parameters were as follows: spray voltage, 3.50 kV; sheath gas rate (N2), 35 L ⋅ h-1; auxiliary gas rate (N2), 10 L ⋅ h-1; capillary temperature, 320°C; and auxiliary gas heater temperature, 350°C. The Orbitrap mass analyzer scanned over a range of m/z 100 to 1500. All the data were recorded and processed by Thermo Scientific Xcalibur 3.0 software (Thermo Fisher Scientific).
High-accuracy MS data were acquired in the negative mode by Full MS/dd-MS2. The HESI source parameters were as follows: spray voltage, 3.50 kV; sheath gas rate (N2), 35 L ⋅ h-1; auxiliary gas rate (N2), 10 L ⋅ h-1; capillary temperature, 320°C; and auxiliary gas heater temperature, 350°C. The Orbitrap mass analyzer scanned over a range of m/z 100 to 1500. All the data were recorded and processed by Thermo Scientific Xcalibur 3.0 software (Thermo Fisher Scientific).
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