Trichloroacetic acid was added to cell culture supernatants (162.8 µl, 72% to 1 ml of supernatant) for protein precipitation. Trp and Kyn concentrations were measured on a Dionex Ultimate® 3000 uHPLC (Thermo Fisher Scientific). Chromatographic separation was achieved on a reversed phase Accucore™ aQ column (2.6 µm, Thermo Scientific) with a gradient mobile phase (see Table S1) consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). Trp and Kyn peaks were identified based on comparison to standards, their retention time and UV emission spectra at 280 nm (Trp) and 365 nm (Kyn). Results were analyzed using the Chromeleon™ 7.2 Chromatography Data System (Thermo Scientific).
Accucore aq column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Accucore™ aQ column is a high-performance liquid chromatography (HPLC) column designed for the analysis of polar and hydrophilic compounds. It features a core-shell particle technology that provides efficient separations and high-quality results.
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4 protocols using accucore aq column
1
Quantifying Tryptophan and Kynurenine
2
Quantitative CJAP Plasma Peptide Analysis
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The Q. Exactive Orbitrap HR-LC-MS/MS was applied to analyze the obtained CJAP plasma treatment peptide mixtures. An Ultimate 3000 UPLC system equipped with an Accucore aQ column (2.1 × 150 mm, 2.6 μm Thermo Scientific, Waltham, MA, USA) was coupled to the quadrupole electrostatic field orbital trap mass spectrometer (Q Exactive, Thermo Scientific, Waltham, MA, USA) in positive mode. The ESI pattern was used as ion source with a scanning range from 50 to 2400 m/z. Solvent A and solvent B consisted of an aqueous solution of 0.1% formic acid in water and of 0.1% formic acid in acetonitrile, respectively. A gradient of acetonitrile from 99% solvent A to 99% solvent B was generated for 20 min at a flow rate of 0.2 mL/min. The Xcalibur software (Thermo Scientific, Waltham, MA, USA) was applied for analysis of MS result.
3
Serum Tryptophan and Kynurenine Analysis by HPLC
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For protein precipitation each 300 μl serum sample was treated with 50.6 μl of 72% trichloroacetic acid. Samples were centrifuged at maximum speed for 12 min and the supernatants were frozen overnight at −20 degrees. After thawing the samples, the centrifugation step was repeated and the obtained supernatants were used for HPLC analysis. A Dionex Ultimate® 3000 uHPLC (Thermo Scientific, Waltham, MA, USA) was used for chromatographic separation of kyn and trp. This was achieved on a reversed phase Accucore™ aQ column (Thermo Scientific™) with 2.6 μm particle size with a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). Kyn and trp were detected based on comparison with standards, their retention times and UV emission spectra at 365 nm and 280 nm, respectively. Results were analyzed using the Chromeleon™ 7.2 Chromatography Data System (Thermo Scientific™ Dionex™).
4
Plasma and Urine Metabolite Analysis
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Thermo Q Exactive UHMR Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Thermo Accucore aQ column (150 mm × 2.1 mm, 2.6 μm) was used to analyze metabolites in plasma and urine. The samples were eluted with 0.1% formic acid water (v/v, solvent A) and acetonitrile (solvent B) as follows: 10–30% B from 0 to 5 min, 30–50% B from 5 to 10 min, 50–70% B from 10 to 20 min, 70–100% B from 20 to 30 min, 100% B from 30 to 35 min and 100–10% B from 35 to 37 min. The column temperature was held at 30 °C; the injection volume was 5 μL, and the flow rate was 0.3 mL/min. Mass spectra were obtained under the following conditions: range, m/z 100–1200; positive and negative ion modes; the collision energy was 20 and 40 eV. Compound Discoverer 3.1 software was used for data analysis.
A total of 100 μL plasma plus 10 μL formic acid vortexed for 1 min, then 300 μL acetonitrile was added and vortexed for 3 min. A total of 1 mL urine plus 50 μL formic acid was vortexed for 1 min; then, 8 mL ethyl acetate was added and vortexed for 3 min. The supernatant was obtained by centrifugation at 5000× g for 10 min at 4 °C and concentrated to dryness by nitrogen blowing. The dry residues were dissolved in 200 μL methanol.
A total of 100 μL plasma plus 10 μL formic acid vortexed for 1 min, then 300 μL acetonitrile was added and vortexed for 3 min. A total of 1 mL urine plus 50 μL formic acid was vortexed for 1 min; then, 8 mL ethyl acetate was added and vortexed for 3 min. The supernatant was obtained by centrifugation at 5000× g for 10 min at 4 °C and concentrated to dryness by nitrogen blowing. The dry residues were dissolved in 200 μL methanol.
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