Cells were extracted in 500 μl of methanol:water (8:2, v:v) containing an internal standard (100 nM acetyl-1,2-13C2-CoA). Samples were sonicated and centrifuged. The supernatant was transferred to new tubes and evaporated. The samples were reconstituted in 100 μl of initial mobile phase (20 mM ammonium acetate in water). The reconstituted solution was filtered, and an aliquot (20 μl) was injected into the LC–MS system. Metabolites were analyzed using an Agilent 1290 Ultra-Performance Liquid Chromatography system coupled with an Agilent 6490 triple quadrupole mass spectrometer (Agilent LC-QQQ-MS/MS, Agilent Technologies) in positive electrospray ionization (ESI) mode. The system was operated in multiple reaction monitoring mode using individually optimized collision energy. Chromatographic separation was achieved on an Agilent Poroshell EC-C18 column (100 mm × 2.1 mm, 2.7 μm). The data processing for both qualitative and quantitative analyses was performed using Agilent MassHunter software (Agilent Technologies).
Agilent 6490 triple quadrupole mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6490 triple quadrupole mass spectrometer is a laboratory instrument designed for precise and sensitive quantitative analysis. It utilizes triple quadrupole technology to provide high-performance mass analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 1260 infinity lc system, by Agilent Technologies (3 mentions)
Masshunter software, by Agilent Technologies (3 mentions)
1260 infinity hplc system, by Agilent Technologies (2 mentions)
Qiaamp fast dna tissue kit, by Qiagen (2 mentions)
Uplc zorbax eclipse plus c18 rrhd column, by Agilent Technologies (2 mentions)
Dna degradase plus, by Zymo Research (2 mentions)
Agilent 1290 lc system, by Agilent Technologies (2 mentions)
Agilent 1290 uplc, by Agilent Technologies (2 mentions)
Acquity beh c18 column, by Waters Corporation (2 mentions)
Agilent 1290 liquid chromatograph, by Agilent Technologies (2 mentions)
Agilent 1290 ultra performance liquid chromatography system, by Agilent Technologies (1 mentions)
Poroshell ec c18 column, by Agilent Technologies (1 mentions)
Agilent mass hunter software, by Agilent Technologies (1 mentions)
Acquity uplc beh amide column, by Waters Corporation (1 mentions)
1260 infinity lc system, by Agilent Technologies (1 mentions)
Hypersil gold column, by Thermo Fisher Scientific (1 mentions)
1290 infinity, by Agilent Technologies (1 mentions)
Agilent 6495 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
High performance liquid chromatography system, by Shimadzu (1 mentions)
33 protocols using agilent 6490 triple quadrupole mass spectrometer
1
Targeted Metabolomics Analysis by LC-MS/MS
2
Quantifying Methamphetamine in Brain Tissue
3
Quantification of Purine Metabolites
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Calibration standards and quality control samples were prepared by serially diluting the stock solution with 50% acetonitrile (ACN). The final eight‐point calibration curve ranges of hypoxanthine, xanthine, and uric acid were 0.5–100, 0.5–100, and 10–2000 μg/mL, respectively. For sample preparation, uric acid‐2‐13C,1,3,7‐15 N3 was used as the internal standard (IS). The IS (50 μg/mL) was added to 20 μL of each urine sample, and the mixture was then extracted using 200 μL of 50% ACN. Next, the mixture was vortexed at room temperature for 5 min and then centrifuged at 12,000 g at 4°C for 10 min. Finally, 100 μL of supernatant was taken into an HPLC vial and 5 μL was injected into a liquid chromatography‐mass spectrometry/mass spectrometry (LC–MS/MS) system. An ACQUITY UPLC BEH Amide Column (2.1 × 50 mm, 1.7 μm; Waters, Milford, MA, USA) was connected to an Agilent 1260 Infinity LC system equipped with an Agilent 6490 triple‐quadrupole mass spectrometer (Santa Clara, CA, USA) for separation and detection of the analytes. The mobile phase consisted of solvents A (0.1% formic acid in HPLC‐grade water) and B (0.1% formic acid in ACN). The mobile phase flow rate was 0.5 mL/min. Multiple reaction monitoring (MRM) was performed in negative electrospray ionization mode.
4
UPLC-QqQ MS for Lipid Profiling
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For the quantitative profiling of lipids, sequential UPLC-QqQ MS analysis was constructed using MRM. The UPLC (1290 infinity, Agilent Technologies, USA) system was composed of a binary pump (G4220A, USA), an autosampler (G4226A, USA), and a column compartment (G1316C, USA). Samples were injected into the sample loop and separated by a Hypersil GOLD column (2.1 × 100 mm, 1.9 μm, Thermo Science) with a linear gradient of solvents A (19 : 19 : 2 acetonitrile : methanol : water, 20 mM ammonium formate, 0.1% formic acid) and B (2-propanol, 20 mM ammonium formate, 0.1% formic acid). The temperatures of the sampler and column oven were set to 4 and 40 °C, respectively. A 33 min gradient was performed as follows: 0–5 min with 5% B, 5–15 min with 5–30% B, 15–22 min with 30–90% B, 22–27 min with 90% B, 27–28 min with 90–5% B, and 28–33 min with 5% B. The flow rate, 250 μL min−1, was adjusted for all gradient times and the injection volume for each run was 4 μL. For MS analysis, an Agilent 6490 Triple Quadrupole mass spectrometer (Agilent Technologies, USA) was used, with the following parameters: 4000 V positive mode capillary voltage, a sheath gas flow of 11 L min−1 (ultra high purity nitrogen), a drying gas flow of 15 L min−1 at various temperatures, and a nebulizer gas flow at 25 psi. Optimized MRM conditions were used to analyze the various lipid species.
5
Targeted Proteomics Workflow Optimization
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Sample block randomization
was performed for each set. The six most abundant proteins were removed
from the samples by a Multiple Affinity Removal System Human-6 (MARS
Hu-6 × 100 mm; Agilent Technologies, Santa Clara, CA) column
that was loaded onto a high-performance liquid chromatography system
(Shimadzu Co., Kyoto, Japan), wherein the column was exchanged for
every 200 samples that were depleted. A total of 100 μg of proteins
from each sample was hydrolyzed with sequencing-grade modified trypsin
(Promega, Madison, WI). The MRM-MS assays on the training and test
sets were conducted on an Agilent 6490 triple quadrupole mass spectrometer
(Agilent Technologies) that was equipped with a Jet Stream Electrospray
source that was coupled to a 1260 Infinity HPLC system (Agilent Technologies),
and the assay on the confirmation set was performed on an Agilent
6495 triple quadrupole mass spectrometer (Agilent Technologies) that
was coupled to the same HPLC system.
Detailed information on
the MRM-MS procedure is provided inSupplementary Methods .
was performed for each set. The six most abundant proteins were removed
from the samples by a Multiple Affinity Removal System Human-6 (MARS
Hu-6 × 100 mm; Agilent Technologies, Santa Clara, CA) column
that was loaded onto a high-performance liquid chromatography system
(Shimadzu Co., Kyoto, Japan), wherein the column was exchanged for
every 200 samples that were depleted. A total of 100 μg of proteins
from each sample was hydrolyzed with sequencing-grade modified trypsin
(Promega, Madison, WI). The MRM-MS assays on the training and test
sets were conducted on an Agilent 6490 triple quadrupole mass spectrometer
(Agilent Technologies) that was equipped with a Jet Stream Electrospray
source that was coupled to a 1260 Infinity HPLC system (Agilent Technologies),
and the assay on the confirmation set was performed on an Agilent
6495 triple quadrupole mass spectrometer (Agilent Technologies) that
was coupled to the same HPLC system.
Detailed information on
the MRM-MS procedure is provided in
6
Quantification of DNA Adducts by LC-MS/MS
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Caecal tissue was harvested as described before and genomic DNA was extracted using QIAamp Fast DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions. 25μg of isolated DNA were spiked with stable isotopically labeled adduct standards and hydrolyzed to 2’-deoxynucleosides (dN) according to our optimized protocol46 (link). After analyte enrichment by C18 solid phase extraction, the predominant DNA adducts after 1-MIM-OH exposure, namely N2-(1-MIM)-dG and N6-(1-MIM)-dA, were analyzed by isotope-dilution liquid chromatography tandem-mass spectrometry (LC-MS/MS) as described previously47 (link) using an Agilent 1260 Infinity LC system coupled to an Agilent 6490 triple quadrupole-mass spectrometer (Agilent Technologies) interfaced with an electrospray ion source operating in the positive ion mode (ESI+). Further, in the DNA hydrolyzate prior to sample purification, we analyzed the amounts of dC and 5mdC (5-methyl-2’-deoxycytidine) by isotope-dilution LC-MS/MS as recently published48 (link). We calculated the amount of DNA that was actually used for the adduct analysis knowing that dC (in fact the sum of dC and 5mdC) accounts for 21% of the total dN in the mouse genome.
7
Quantification of DNA Adducts by LC-MS/MS
8
Quantification of TMAO, Choline, and Betaine
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Plasma heparin samples were obtained after a 12 h fast and stored at -80 °C. Quantification of TMAO, choline and betaine was performed by LC–MS/MS as done by Missailidis29 (link), utilizing a protocol designed specifically for this purpose and prepared in a 96-well format. Extracted plasma aliquots were spiked with internal standards, comprised of TMAO-D9 in methanol and water with Proline-13C5 as a recovery standard, and injected on an Agilent 1290 Infinity chromatographic system (Agilent Technologies, Waldbronn, Germany) fitted with an Acquity UPLC Amide column in combination with a VanGuard precolumn (Waters Corporation, Milford, MA, USA). The compounds were detected with an Agilent 6490 Triple Quadrupole mass spectrometer (Agilent Techologies, Santa Clara, CA, USA). Data processing was performed with MassHunter Quantitative Analysis QQQ (Agilent Technologies Inc. Santa Clara, CA, USA). The MS/MS analyses for TMAO, choline, betaine, TMAO- D9 and Proline-13C5, were conducted in multiple-reaction-monitoring (MRM) mode at m/z 76 → 58, m/z 104 → 45, m/z 118 → 58, m/z 85 → 66 and m/z 121 → 74 respectively.
9
Quantitative Analysis of DNA Modifications
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Genomic DNA was hydrolyzed by DNA Degradase Plus (Zymo Research) according to the manufacturer's instructions. Digested DNA was injected onto a UPLC Zorbax Eclipse Plus C18 RRHD column (Agilent Technologies). The analytes were separated by gradient elution using 5% methanol/0.1% formic acid (mobile phase A) and 100% methanol (mobile phase B) at a flow rate of 0.25 mL/min. Mobile phase B was increased from 0 to 3% in 5 min, to 80% in 0.5 min, kept at 80% for 2 min, then switched to initial conditions in 2.5 min. The effluent from the column was directed to the Agilent 6490 Triple Quadrupole mass spectrometer (Agilent Technologies). The following transitions were monitored: m/z 228.1 → 112.1 (C); m/z 242.1 → 126.1 (5-mC); and m/z 258.1 → 142.1 (5-hmC).
Calibration solutions with varying amounts of 5-hmC (0%–3%), 5-mC (0%–10%), and fixed amount of C, were also analyzed together with the samples. The solutions were prepared from a 200-bp DNA standard containing 57 cytosines, which are homogeneous for C, 5-hmC, or 5-mC. Calibration plots of %5-hmC or %5-mC versus MRM Response ratio were constructed based on the data obtained, and %5-hmC is obtained from the ratio of [5-hmC/(5-mC+5-hmC+C)]. Response ratio is the response peak area for 5-hmC or 5-mC divided by the combined peak areas of 5-hmC, 5-mC, and C. The %5-hmC or 5-mC in the samples was determined from the calibration plots.
Calibration solutions with varying amounts of 5-hmC (0%–3%), 5-mC (0%–10%), and fixed amount of C, were also analyzed together with the samples. The solutions were prepared from a 200-bp DNA standard containing 57 cytosines, which are homogeneous for C, 5-hmC, or 5-mC. Calibration plots of %5-hmC or %5-mC versus MRM Response ratio were constructed based on the data obtained, and %5-hmC is obtained from the ratio of [5-hmC/(5-mC+5-hmC+C)]. Response ratio is the response peak area for 5-hmC or 5-mC divided by the combined peak areas of 5-hmC, 5-mC, and C. The %5-hmC or 5-mC in the samples was determined from the calibration plots.
10
Quantifying DNA Modifications: LC-MS/MS Method
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Genomic DNA was hydrolyzed by DNA Degradase Plus (Zymo Research, CA, USA) according to the manufacturer’s instructions. Digested DNA was injected onto a UPLC Zorbax Eclipse Plus C18 RRHD column (Agilent Technologies, CA). The analytes were separated by gradient elution using 5% methanol/0.1% formic acid (mobile phase A) and 100% methanol (mobile phase B) at a flow rate of 0.25 ml/min. Mobile phase B was increased from 0% to 3% in 5 min, to 80% in 0.5 min, kept at 80% for 2 min then switched to initial conditions in 2.5 min. The effluent from the column was directed to the Agilent 6490 Triple Quadrupole mass spectrometer (Agilent Technologies, CA). The following transitions were monitored: m/z 228.1 - > 112.1 (C); m/z 242.1- > 126.1 (5mC) and m/z 258.1- > 142.1 (5hmC).
Calibration solutions with varying amounts of 5hmC (0–3%), 5mC (0–10%) and fixed amount of C, were also analyzed together with the samples. The solutions were prepared from a 200 bp DNA standards containing 57 cytosines which are homogeneous for C, 5hmC or 5mC. Calibration plots of % 5hmC or %5mC vs MRM Response ratio were constructed based on the data obtained. %5hmC is obtained from the ratio of [5hmC]/5hmC]+[C]. Response ratio is the response peak area for 5hmC or 5mC divided by the combined peak areas of 5hmC, 5mC and C. The % 5hmC or 5mC in the samples were determined from the calibration plots.
Calibration solutions with varying amounts of 5hmC (0–3%), 5mC (0–10%) and fixed amount of C, were also analyzed together with the samples. The solutions were prepared from a 200 bp DNA standards containing 57 cytosines which are homogeneous for C, 5hmC or 5mC. Calibration plots of % 5hmC or %5mC vs MRM Response ratio were constructed based on the data obtained. %5hmC is obtained from the ratio of [5hmC]/5hmC]+[C]. Response ratio is the response peak area for 5hmC or 5mC divided by the combined peak areas of 5hmC, 5mC and C. The % 5hmC or 5mC in the samples were determined from the calibration plots.
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