Fecal samples collected from the mice and tissues collected from the CRC patients were mixed with pre-cooled methanol/acetonitrile/water solution in the ratio of 2:2:1 (v/v), followed by vortex mixing, low-temperature ultrasonic processing for 30 min, freezing at −24°C for 10 min, and then centrifugation at 14,000 rpm for 20 min at 4°C. Later, the supernatant was extracted and dried in vacuum. Subsequently, 100 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v/v) was added for mass spectrometry, followed by vortex mixing and centrifugation at 14,000 rpm at 4°C for 15 min. Finally, the samples were sent with clear liquid for analysis by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China). Mouse feces and human tissues were detected by Agilent 7890B/7000D GC-MS (Agilent, United States) for targeted detection of SCFA. In addition, mouse feces were also tested for untargeted metabolites by UHPLC-Q-TOF MS (Agilent, United States; AB SCIEX, United States).
Uhplc q tof ms
Manufactured by Agilent Technologies
Sourced in United States
The UHPLC-Q-TOF-MS is a high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry system. It is designed for the separation, identification, and quantification of complex chemical compounds with high accuracy and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Modular p800 biochemical analyzer, by Roche (2 mentions)
Bt225 electronic analytical balance, by Sartorius (2 mentions)
Acquity uplc beh, by Waters Corporation (2 mentions)
Agilent 6540, by Agilent Technologies (2 mentions)
Agilent 7890b 7000d gc ms, by Agilent Technologies (1 mentions)
Uplc beh amide column, by Waters Corporation (1 mentions)
Milli q advantage a10 ultra pure water purifier, by Merck Group (1 mentions)
Lc ms grade, by Avantor (1 mentions)
Pegasus 4d gc gc tofms instrument, by Leco (1 mentions)
Uplc c18 analytical column, by Waters Corporation (1 mentions)
Van guard beh, by Waters Corporation (1 mentions)
Ab triple tof 6600 mass spectrometer, by AB Sciex (1 mentions)
1290 infinity ultrahigh performance liquid chromatography system, by Agilent Technologies (1 mentions)
Acquity hss t3 column, by Waters Corporation (1 mentions)
Simca p 14, by Sartorius (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
15 protocols using uhplc q tof ms
1
Untargeted and Targeted Metabolomic Analysis of Mouse Feces and Human Colorectal Cancer Tissues
2
Rice Metabolite Extraction and Analysis
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The metabolites of rice were extracted and analyzed by the method described previously (Zhang Y. et al., 2017 (link)). Briefly, a total of 50 mg of fresh leaves was ground in liquid nitrogen, and transferred into the new EP tube containing 1 mL of an internal target substance (Vmethanol:Vacetonitrile:Vwater = 2:2:1, which was kept at -20°C in advance), and homogenized by ball mill at 45 Hz for 4 min, then treated on ice bath by continuous three times of ultrasound for 5 min, then incubated at -20°C for 1 h to precipitate proteins, then centrifuged by 14500 × g at 4°C for 15 min. A total of 500 μL of supernatants were transferred into the new EP tubes, and dried in a vacuum concentrator without heating, and was supplemented by a reconstitution with 100 μL of extraction solution (Vacetonitrile:Vwater = 1:1), and mixed for 30 sec, and sonicated in a water bath at 4°C for 10 min, and centrifuged by 14500 × g at 4°C for 15 min. The supernatants were filtered through the PTFE membrane of 0.22 mm (Sigma, United States), and subjected to analyses on the UHPLC-QTOF-MS (1290, Agilent Technologies) equipped with a UPLC BEH Amide column (1.7 μm, 2.1 mm × 100 mm, Waters Corporation, United States) using optimized mobile phases. The extraction and analysis of metabolites were assisted by the Allwegene Technology Co., Ltd. (Beijing, China).
3
Modular Biochemical Analysis Platform
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Modular p800 Biochemical analyzer (Roche, Germany), high-speed refrigeration centrifuge (Thermo Scientific, Germany), BT225-Electronic analytical balance (Sartorius, Germany), SpeedVac® SPD131Centrifuge enrichment system (Thermo Scientific, Germany), Milli-Q Advantage A10 Ultra-pure Water Purifier (Millipore, USA), Agilent 6538A ultrahigh-performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) (Agilent, USA).
4
Untargeted Metabolomics Analysis of Grape Juice
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The untargeted metabolomics
analysis was directly conducted on grape juice following centrifugation
(8000g for 15 min) and filtration (cellulose membrane,
0.22 μm) into amber glass vials. Analysis was done using ultra-high-pressure
liquid chromatography coupled to a quadrupole-time-of-flight mass
spectrometer (UHPLC/QTOF-MS) from Agilent Technologies (Santa Clara,
CA, USA), as previously reported.29 (link) Briefly,
the chromatographic separation was achieved using a Pursuit 3 pentafluorophenyl
(PFP) column (2.0 × 100 mm, 3 μm) from Agilent Technologies
(Santa Clara, CA, USA) and a binary gradient from 6 to 94% acetonitrile
(LC–MS grade, VWR, Milan, Italy) in 33 min with a flow rate
of 200 μL min–1. The samples were injected
randomly, with an injection volume of 6 μL, and SCAN acquisition
was adopted (100–1000 m/z, 30,000 fwhm, 1 Hz). The raw data were processed using Agilent Profinder
B.06 software, as previously described,24 (link) to putatively annotate compounds based on isotopic spacing and ratio
according to the “find-by-formula” algorithm (5 ppm
mass accuracy for monoisotopic mass and 0.05 min for retention time
alignment). The PlantCyc 12.6 database25 (link) was used as a reference, and level 2 confidence was achieved concerning
the COSMOS Metabolomics Standards Initiative.26 (link) The Metabolomics data were deposited into the EMBL-EBI MetaboLights
database with the identifier MTBLS7841.
analysis was directly conducted on grape juice following centrifugation
(8000g for 15 min) and filtration (cellulose membrane,
0.22 μm) into amber glass vials. Analysis was done using ultra-high-pressure
liquid chromatography coupled to a quadrupole-time-of-flight mass
spectrometer (UHPLC/QTOF-MS) from Agilent Technologies (Santa Clara,
CA, USA), as previously reported.29 (link) Briefly,
the chromatographic separation was achieved using a Pursuit 3 pentafluorophenyl
(PFP) column (2.0 × 100 mm, 3 μm) from Agilent Technologies
(Santa Clara, CA, USA) and a binary gradient from 6 to 94% acetonitrile
(LC–MS grade, VWR, Milan, Italy) in 33 min with a flow rate
of 200 μL min–1. The samples were injected
randomly, with an injection volume of 6 μL, and SCAN acquisition
was adopted (100–1000 m/z, 30,000 fwhm, 1 Hz). The raw data were processed using Agilent Profinder
B.06 software, as previously described,24 (link) to putatively annotate compounds based on isotopic spacing and ratio
according to the “find-by-formula” algorithm (5 ppm
mass accuracy for monoisotopic mass and 0.05 min for retention time
alignment). The PlantCyc 12.6 database25 (link) was used as a reference, and level 2 confidence was achieved concerning
the COSMOS Metabolomics Standards Initiative.26 (link) The Metabolomics data were deposited into the EMBL-EBI MetaboLights
database with the identifier MTBLS7841.
5
UHPLC-Q/TOF-MS Analysis of Chemical Compounds
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The analysis was performed on an Agilent 6540 ultrahigh definition accurate mass quadrupole time-of-flight spectrometer with UHPLC (UHPLC-Q/TOF-MS; Agilent Technologies, Santa Clara, CA, USA). A UHPLC C18 column (2.1 mm × 100 mm, inner diameter 1.7 μm, Acquity UPLC BEH; Waters, Milford, MA, USA) with a C18 pre-column (2.1 mm × 5 mm, inner diameter 1.7μm, VanGuard BEH; Waters) was used for separation at 20°C. The mobile phase consisted of 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B). The elution condition was optimized as follows: 0–3 min, 10–20 % B; 3–25 min, 20–38 % B; 25–30 min, 38–85 % B; 30–30.1 min, 85–100 % B. The flow rate was at 0.35 mL/min, and the injection volume was 1 μL.
Mass spectrometry was performed in negative mode with the mass to charge ratio (m/z) ranging from 100 to 1700. Dry gas (N2) was set to 8 L/min at 300°C. Nebulizer pressure was 310264 Pa, and Vcap was 3000 V. Nozzle voltage and fragmentor voltage were set at 500 V and 180 V respectively.
Mass spectrometry was performed in negative mode with the mass to charge ratio (m/z) ranging from 100 to 1700. Dry gas (N2) was set to 8 L/min at 300°C. Nebulizer pressure was 310264 Pa, and Vcap was 3000 V. Nozzle voltage and fragmentor voltage were set at 500 V and 180 V respectively.
6
Metabolomic and Lipidomic Profiling of Serum
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The serum samples were immediately stored at − 80 °C until analysis in 2017. The methods used for analyzing metabolomics and lipidomics have been described in previous papers [8 (link), 11 (link)]. In brief, a non-targeted two-dimensional gas chromatography time-of-flight mass-spectrometry method Leco Pegasus 4D GC × GC-TOFMS instrument (Leco Corp., MI, USA) was used for the metabolomics analysis which subsequently identified 75 metabolites included in the data analysis. The lipidomics analysis was performed with an ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry method UHPLC-QTOF/MS (Agilent Technologies, Santa Clara, CA, USA) [12 (link), 13 (link)] identifying 106 named molecular lipid species. Metabolites or molecular lipid species with equal to or less than 20% missing or undetectable values were included in the analysis and all missing values were imputed with the k-nearest neighbor algorithm. Finally, all values were log2-transformed [14 (link)].
7
UHPLC-QTOF-MS Analysis of Metabolites
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UHPLC–QTOF-MS analysis was performed on an Agilent 6540 ultra-high definition accurate mass quadrupole time-of-flight spec-trometer with UHPLC (UHPLC–QTOF-MS, Agilent Technologies, U.S.A.). A UPLC C18 analytical column (2.1 mm × 100 mm, I.D. 1.7 μm, ACQUITY UPLC®BEH, Waters, U.S.A.) was used for separation, coupled with a C18 pre-column (2.1 mm × 5 mm, I.D. 1.7 μm, Van-GuardTM BEH, Waters, U.S.A.) at room temperature of 20 °C. The mobile phase was a mixture of water (A) and acetonitrile (B), both containing 0.1% formic acid, with an optimized linear gradient elution as follows: 0–5 min, 10–35% B; 5–25 min, 35–55% B; 25–28 min, 55–85% B; 28–30 min, 85–100% B. The injection volume was 4 μL. The flow rate was set at 0.35 mL/min. The mass spectra were acquired in negative mode by scanning from 100 to 1700 in mass to charge ratio (m/z). The MS analysis was performed under the following operation parameters: dry gas temperature 300 °C, dry gas (N2) flow rate 5 L/min, nebulizer pressure 30 psi, Vcap 3000, nozzle voltage 500 V, and fragmentor voltage 200 V.
8
Untargeted Lipidomic Profiling of Fasting Serum
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Fasting serum samples were analyzed in a random order in positive and negative electrospray ionization modes using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) (Agilent Technologies, Santa Clara, CA, USA) [17 (link),18 (link),19 (link),20 (link),21 (link),22 (link)] (Supplementary Methods ). A total of 279 lipids were semiquantified into the following lipid classes: triglycerides (TG), phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), lysophosphatidylcholines (LPC), sphingolipids (SM), cholesteryl esters (CE), and ceramides (CER) (Table S2 ).
9
Metabolomic Analysis of MitoQ-Treated Cells
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The cells were treated using MitoQ, and after collection, the cells were washed with pre-cooled phosphate buffered saline (PBS) three times and removed from the supernatant. Pre-cooled methanol: acetonitrile: water (2:2:1, v/v) solution was added to each sample to precipitate protein. The samples were pulverized by the ultrasonic wave at 0 °C for 30 min, incubated at −20 °C for 10 min, and then centrifuged at 4 °C, 14,000× g, for 20 min. The supernatants were transferred into new 1.5 mL tubes and dried inside a centrifugal vacuum evaporator, then reconstituted using a 100 μL acetonitrile: water (1:1, v/v) mixture for further analysis. The metabolites in the samples were detected with Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China) using UHPLC-Q-TOF MS. which is a 1290 Infinity Ultra-high Performance Liquid Chromatography system (Agilent, Santa Clara, CA, USA) coupled with AB Triple TOF 6600 Mass Spectrometer (AB SCIEX, Radio Road Redwood City, CA, USA).
10
Non-targeted Metabolomic Analysis of Yellow Teas
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The non-targeted metabolomic analysis of yellow teas was performed on an ultrahigh performance liquid chromatography–quadrupole-time-of-flight mass spectrometry (UHPLC–QTOF-MS; Agilent Technologies Inc., Santa Clara, California, USA) equipped with an ACQUITY HSS T3 column (1.8 mm, 2.1 mm × 100 mm; Waters, Milford, Massachusetts, USA), as described previously (Zhu et al., 2017 (link), Zhu et al., 2021 (link)). The metabolite profiles were analyzed by using the Mass Profiler Professional 13.0 software (Agilent Technologies Inc., Santa Clara, California, USA).
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