A high-performance Q-Exactive (ThermoScientific) mass spectrometer featuring quadrupole precursor selection with high resolution, accurate-mass (HR/AM) orbitrap detection was used for all MS analyses. The Accela ultra high-performance liquid chromatography system (ThermoScientific) was used for LC separation experiments which also features a photodiode array detector for absorbance measurements across multiple wavelengths. Measurements were carried out under ambient conditions and employed a capillary inlet temperature of 250 °C. A Model Fusion 200 syringe pump from Chemyx Inc was used to pump analytes at set flow rates.
Accela ultra high performance liquid chromatography system
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Accela ultra-high-performance liquid chromatography (UHPLC) system is a laboratory instrument designed for the separation, identification, and quantification of complex chemical mixtures. It utilizes high-pressure liquid chromatography techniques to achieve rapid and efficient separation of analytes. The Accela UHPLC system is capable of operating at pressures up to 1,000 bar, enabling the use of smaller particle size columns and achieving improved resolution, sensitivity, and speed compared to traditional HPLC systems.
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Lab products found in correlation
Q exactive, by Thermo Fisher Scientific (1 mentions)
Lys c, by Roche (1 mentions)
Rapigest, by Waters Corporation (1 mentions)
Trypsin, by Roche (1 mentions)
Luna 5 micron c18 column, by Phenomenex (1 mentions)
600 mhz spectrometer, by Agilent Technologies (1 mentions)
Hypersil gold reversed phase c18 column, by Thermo Fisher Scientific (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Vacuum pump v 700, by Büchi (1 mentions)
Vacuum controller v 850, by Büchi (1 mentions)
Tsq quantum access triple quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Rotavapor r 210, by Büchi (1 mentions)
Hypersil gold column, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
6 protocols using accela ultra high performance liquid chromatography system
1
High-Resolution MS for Liquid Chromatography
2
FFPE Tissue Proteome Extraction and Quantification
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FFPE tissue slices (20 μm thick) were deparaffinized in xylenes/ethanol, and proteins extracted at 95°C × 20 min in 150 mM NaCl, 50 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylenediaminetetraacetic acid (EDTA), 4% Rapigest (Waters Corp., Milford, MA), pH 7.4, and then digested at 80°C × 2 h in 150 mM NaCl, 50 mM HEPES, 1 mM EDTA, pH 7.4. Samples were sequentially deglycosylated (EMD Chemicals, Cambridge, MA), proteolyzed (Lys-C and trypsin, Roche Applied Science), and reduced in Tris(2-carboxyethyl)phosphine. Protein yields were determined before and after digestions using a Bio-Rad DC Protein Assay kit (Life Science, Hercules, CA). Liquid chromatography was performed on an Accela ultra-high-performance liquid chromatography system (Thermo Fisher Scientific, Waltham, MA) and multiple reaction monitoring (MRM) mass spectrometry on a TSQ Vantage quadrupole mass spectrometer (Thermo Fisher Scientific) with 30 min acquisition time. Peptide spectra were generated in triplicate and analyzed using Skyline freeware (University of Washington, Seattle, WA).
3
Enzyme-Catalyzed Metabolite Synthesis and Purification
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Frozen cell paste was thawed at room temperature
(rt) and resuspended
in 20 mL (50 mg/mL) of AO buffer (pH 7.4) in a 50 mL centrifuge tube.
The cell paste suspension was prewarmed for 5 min (37 °C); then,
100 μL of the substrate (zoniporide, 2 mg; DACA, 4 mg; zaleplon,
2 mg; O6BG, 2 mg) dissolved in DMSO was added. The incubation
was performed at 37 °C with replacement of cell paste when substrate
conversion slowed, as monitored by LC–MS/MS. Once the reaction
had advanced to completion (about 5 days), the cells were spun down
at 30 000g for 20 min. The supernatant was
extracted with ethyl acetate. The organic layer was collected and
concentrated in vacuo. Further purification was performed on an Accela
ultra high-performance liquid chromatography system (Thermo Scientific,
Waltham, MA) with a 4.60 × 250 mm2 Luna 5 μm
C18 column (Phenomenex, Torrance, CA). Mobile phases A and B were
identical to those used for mass spectrometry. The column was equilibrated
with 90% mobile phase A (4.0 min; 800 μL/min). Chromatographic
separation was achieved using a linear gradient for over 13 min to
0% mobile phase A. The purified metabolites were identified using
LC–MS/MS and proton NMR. NMR spectra (seeSupporting Information ) were recorded using a Varian 600 MHz
spectrometer.
(rt) and resuspended
in 20 mL (50 mg/mL) of AO buffer (pH 7.4) in a 50 mL centrifuge tube.
The cell paste suspension was prewarmed for 5 min (37 °C); then,
100 μL of the substrate (zoniporide, 2 mg; DACA, 4 mg; zaleplon,
2 mg; O6BG, 2 mg) dissolved in DMSO was added. The incubation
was performed at 37 °C with replacement of cell paste when substrate
conversion slowed, as monitored by LC–MS/MS. Once the reaction
had advanced to completion (about 5 days), the cells were spun down
at 30 000g for 20 min. The supernatant was
extracted with ethyl acetate. The organic layer was collected and
concentrated in vacuo. Further purification was performed on an Accela
ultra high-performance liquid chromatography system (Thermo Scientific,
Waltham, MA) with a 4.60 × 250 mm2 Luna 5 μm
C18 column (Phenomenex, Torrance, CA). Mobile phases A and B were
identical to those used for mass spectrometry. The column was equilibrated
with 90% mobile phase A (4.0 min; 800 μL/min). Chromatographic
separation was achieved using a linear gradient for over 13 min to
0% mobile phase A. The purified metabolites were identified using
LC–MS/MS and proton NMR. NMR spectra (see
spectrometer.
4
Serum Sample Preparation and HPLC Analysis
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The serum sample was thawed, and 100 μL was collected. This aliquot was mixed with 300 μL acetonitrile to form a volume ratio of 1:3. After vigorous shaking for 30 seconds, followed by centrifugation at 12,000 g for 5 minutes at 4°C, the supernatant was removed and was filtered by a 0.22-μm filter (Millipore) to obtain the sample to be tested. Liquid chromatography was performed using the Accela ultra-high-performance liquid chromatography system from Thermo Fisher Scientific. The chromatographic column was a Thermo Hypersil GOLD reversed phase C18 column (2.1 mm ID×50 mm, 1.9 μm). The two-solvent gradient chromatography elution mode was used. Mobile phase A was 0.1% formic acid aqueous solution. Mobile phase B was 0.1% formic acid acetonitrile solution. The chromatographic elution process was 15 min, the injection volume was 10 μL, and the flow rate was set to 200 μL/min. The auto-sampler temperature was set to 4°C, and the column temperature was set to 20°C. The initial gradient was 5% B and was maintained for 2.5 min. At 8.5 min, the gradient increased to 95%. It was later decreased to 5% B after 3 min of maintenance. After that, 2.5 min was taken to balance the chromatography column.
5
Quantitative Phenolic and Carotenoid Analysis
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Extract condensation was performed under vacuum and heat-assisted evaporation, in temperatures below 35 °C, using a Büchi Rotavapor R-210 apparatus, equipped with a Büchi vacuum pump V-700, Vacuum controller V-850 (all obtained from Büchi, Flawil, St Gallen, Switzerland), and Julabo F12 (Seelbach, Germany) cooling unit.
The estimation of Total Phenolic Content (TPC) and Total Tannin Content (TTC) was implemented using an Infinite® 200 PRO microplate reader (Tecan Group Ltd., San Jose, CA, USA) and the estimation of Total Carotenoid Content (TCC) was performed using an x-ma 100 spectrophotometer (Human Corporation, Seoul, Republic of Korea).
An Accela Ultra High-Performance Liquid Chromatography system equipped with an autosampler and coupled with a TSQ Quantum Access triple-quadrupole mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for the determination of the phenolic and carotenoid compounds qualitative and quantitative content.
The estimation of Total Phenolic Content (TPC) and Total Tannin Content (TTC) was implemented using an Infinite® 200 PRO microplate reader (Tecan Group Ltd., San Jose, CA, USA) and the estimation of Total Carotenoid Content (TCC) was performed using an x-ma 100 spectrophotometer (Human Corporation, Seoul, Republic of Korea).
An Accela Ultra High-Performance Liquid Chromatography system equipped with an autosampler and coupled with a TSQ Quantum Access triple-quadrupole mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for the determination of the phenolic and carotenoid compounds qualitative and quantitative content.
6
Metabolomic analysis by UHPLC-Orbitrap MS
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All tissue extract samples and QC samples were reconstituted in 200µL 50:50 water:methanol prior to analysis. The samples were analysed using an Accela Ultra High Performance Liquid Chromatography system coupled to an electrospray hybrid LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific, Bremen, Germany). All samples were analysed separately in positive and negative ion modes. QC samples were analysed for the first ten injections and then every fifth injection.
The last two injections were also QC samples. Chromatographic separations were performed, following injection of a 10µL sample volume, onto a Hypersil GOLD column (100x2.1mm, 1.9µm; ThermoFisher Scientific, Runcorn, UK) with the column temperature set at 50°C. The two solvents applied w ere solvent A -0.1% formic acid in water (vol/vol) and solvent B -0.1% formic acid in methanol (vol/vol)) at a flow rate of 400µL/min. Solvent A was held at 100% for 0.5 minutes followed by an increase to 100% solvent B over 4.5 minutes, which was then held at 100% solvent B for a further 5.5 minutes. At 10.5 minutes, it was changed to 100% solvent A and held at 100% solvent A to equilibrate for 1.5 minutes. All column eluent was transferred to the mass spectrometer. Full-scan profiling data were acquired in an Orbitrap mass analyser (mass resolution 30,000 at m/z =400).
The last two injections were also QC samples. Chromatographic separations were performed, following injection of a 10µL sample volume, onto a Hypersil GOLD column (100x2.1mm, 1.9µm; ThermoFisher Scientific, Runcorn, UK) with the column temperature set at 50°C. The two solvents applied w ere solvent A -0.1% formic acid in water (vol/vol) and solvent B -0.1% formic acid in methanol (vol/vol)) at a flow rate of 400µL/min. Solvent A was held at 100% for 0.5 minutes followed by an increase to 100% solvent B over 4.5 minutes, which was then held at 100% solvent B for a further 5.5 minutes. At 10.5 minutes, it was changed to 100% solvent A and held at 100% solvent A to equilibrate for 1.5 minutes. All column eluent was transferred to the mass spectrometer. Full-scan profiling data were acquired in an Orbitrap mass analyser (mass resolution 30,000 at m/z =400).
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