Diluted (1:1000 tryptophan and 1:100 for kynurenine) samples (20 µL) were subjected to injection using an Agilent autosampler liquid chromatography–tandem mass spectrometry (LC-MS/MS) with an analytical Waters Symmetry C18, 3.5-µm column connected to the 5500 iontrap (Sciex, Framingham, MA) fitted with a turbo V electrospray source. The samples were subjected to a linear gradient of 2% acetonitrile, 0.1% formic acid to 98% acetonitrile 0.1% formic acid for 10 minutes at a column flow rate of 250 µL/min. Transitions monitored are in Table S1. The data were analyzed using MultiQuant (Applied Biosystems, Foster City, CA), which provided the peak area for the transitions. A standard curve was constructed using concentration ratios of heavy tryptophan/tryptophan and heavy kynurenine/kynurenine from fentomole to nanomole in 20 µL.
Multiquant
Manufactured by Thermo Fisher Scientific
Sourced in United States
MultiQuant is a software application developed by Thermo Fisher Scientific for the analysis and quantification of mass spectrometry data. The core function of MultiQuant is to provide users with a comprehensive platform for processing and interpreting complex analytical data.
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6 protocols using multiquant
1
Quantification of Tryptophan and Kynurenine
2
Targeted Metabolite Profiling by Liquid Chromatography-Mass Spectrometry
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Total ion chromatograms were analyzed using Peakview (version 1.2.0.3, Applied Biosystems). The acquired raw data were imported into Marker View (version 1.2.1.1, Applied Biosystems) for peak extraction and alignment. Features that detected in at least 80% of acquired samples in disease or control group were retained [26 (link)]. Besides, due to the automatic peak alignment process, the information of low abundance of metabolites may be lost. To obtain a comprehensive and accurate profile of BA, we performed a method of targeted extraction and integration of the chromatographic peak using MultiQuant (version 2.1, Applied Biosystems) software (Supplementary Materials
3
Quantitative LC-MS/MS for Tryptophan and Kynurenine
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Diluted samples (20 µL, diluted 1:1000 for Trp and 1:100 for Kyn) were subjected to injection using an Agilent autosampler LC-MS/MS with an analytical Waters Symmetry C18, 3.5 µm column connected to the 5500 iontrap (Sciex, Framingham, Massachusetts, USA) fitted with a turbo V electrospray source. The samples were subjected to a linear gradient of 2% ACN, 0.1% formic acid to 98% ACN 0.1% formic acid for 10 min at a column flow rate of 250 µL/min. Transitions monitored are in online supplementary table 1S . The data were analysed using MultiQuant (Applied Biosystems, Foster City, California, USA) providing the peak area for the transitions. A standard curve was constructed using concentration ratios of Trp/Trp and Kyn /Kyn from fentomole to nanomole in 20 µL.
4
Targeted Metabolomics Analysis via LCMS
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A replicate plate was set up during tracing experiments to assess relative metabolite concentrations using LCMS and harvested as described above. Dried metabolites were reconstituted in 100 μl of 0.03% formic acid in analytical-grade water, vortexed and centrifuged to remove insoluble material. The supernatant was collected and subjected to targeted metabolomics analysis as described on an AB SCIEX QTRAP 5500 liquid chromatography/triple quadrupole mass spectrometer (Applied Biosystems SCIEX) (Kim et al., 2000 (link)). The injection volume was 20 μl. Chromatogram review and peak area integration were performed using MultiQuant (version 2.1, Applied Biosystems SCIEX). The peak area for each detected metabolite was normalized against the total ion count of that sample.
5
Quantification of DHA and EPA in Samples
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Sample extracts were run on an AB SCIEX 3200 triple quadrupole mass spectrometer in negative ionization mode on a Kinetex 2.6 µm C18 100 Å, LC Column 50 × 3 mm. A gradient was used to separate the compounds using mobile phase A: 95:5 water with 0.1% formic acid:methanol and mobile phase B: methanol. Samples were separated using a linear gradient from 10%B to 90%B over 15 min with a flow rate of 0.300 mL/min. Samples (20 µL injections) were analyzed on the LC/MS/MS with a column temperature of 40°C. The mass spectrometer parameters, including the declustering potentials and collision energies, were optimized for DHA (283.2/229.2) and EPA (257.2/203.3). Analyst 1.6.2 (Applied Biosystems) and Multiquant (Applied Biosystems) were used to collect and analyze data. The external standards calibration curve utilized a linear regression model for response ratios (peak area of analyte/peak area of internal standard) to determine the amount of DHA and EPA in the samples. The signal-to-noise ratio (S/N) was used to determine the limit of detection (LOD; S/N ≥ 3) and the limit of quantitation (LOQ; S/N ≥ 10). Difference values for DHA and EPA were calculated between timepoints by subtracting 16-week from 36-week values.
6
Mass Spectrometry Serum Sample Preparation
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Serum samples were prepared for mass spectrometry as described [23] (link). Samples were lyophilized and redissolved in 2% ACN containing 0.1% formic acid, and peaked with 50 fmol of peptide mixture of βgalactosidase, as a relative internal standard peptide for LC-MS/MS analysis as described [24] (link).
MRM experiments were performed on 4000 QTRAP mass spectrometer (Applied Biosystems) interfaced with a 2-D nanoLC (Eksigent) was used to perform LC-MS/MS analysis. MRM data on the 4000 QTRAP mass spectrometer were acquired with NanoSpray II source. The optimal acquisition parameters were as follows: ion spray voltage (2300 V), curtain gas (30 p.s.i.), nebulizer gas (16 p.s.i.), interface heater temperature (150 ℃), declustering potential (100). The resolution parameters of the rst and the third quadrupoles were set as "unit". In the MRM runs, the scan time was maintained at 50ms for each transition, and the pause between transition scans was set to 5ms. Result les (wiff and wiff.scan) were imported into peak area integration software, MultiQuant (Applied Biosystems, version 1.1) to extract the peak areas of transitions and to normalize using the peak area of internal standard peptide for the βgalactosidase peptide (VDEDQPFPAVPK, IDPNAWVER, GDFQFNISR) to adjust for variations between runs, as described.
MRM experiments were performed on 4000 QTRAP mass spectrometer (Applied Biosystems) interfaced with a 2-D nanoLC (Eksigent) was used to perform LC-MS/MS analysis. MRM data on the 4000 QTRAP mass spectrometer were acquired with NanoSpray II source. The optimal acquisition parameters were as follows: ion spray voltage (2300 V), curtain gas (30 p.s.i.), nebulizer gas (16 p.s.i.), interface heater temperature (150 ℃), declustering potential (100). The resolution parameters of the rst and the third quadrupoles were set as "unit". In the MRM runs, the scan time was maintained at 50ms for each transition, and the pause between transition scans was set to 5ms. Result les (wiff and wiff.scan) were imported into peak area integration software, MultiQuant (Applied Biosystems, version 1.1) to extract the peak areas of transitions and to normalize using the peak area of internal standard peptide for the βgalactosidase peptide (VDEDQPFPAVPK, IDPNAWVER, GDFQFNISR) to adjust for variations between runs, as described.
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