Thymine d4

Sources for reagents were: HPLC-grade acetonitrile and water (Burdick & Jackson); mass spectrometry-grade formic acid and ammonium acetate (Sigma-Aldrich); calibration solution containing multiple calibrants in a solution of acetonitrile, trifluroacetic acid, and water (Agilent Technologies); metabolite standards and internal standards, including N-acetyl Aspartic acid-d3, Tryptophan-15N2, Sarcosine-d3, Glutamic acid-d5, Thymine-d4, Gibberellic acid, Trans-Zeatine, Jasmonic acid, 15N Anthranilic acid, and Testosterone-d3 (Sigma-Aldrich).

High-performance liquid chromatography (HPLC) grade acetonitrile, methanol and water were purchased from Burdick & Jackson (Morristown, NJ). Mass spectrometry grade formic acid and internal standards namely, Tryptophan-15N2, Glutamic acid-d5, Thymine-d4, Gibberellic acid, Trans-Zeatin, Jasmonic acid, Anthranilic acid, and Testosterone-d3 were purchased from Sigma- Aldrich (St.Louis, MO). The calibration solution containing multiple calibrants in acetonitrile/trifluroacetic acid /water was purchased from Agilent Technologies (Santa Clara, CA).

High-performance liquid chromatography (HPLC) grade acetonitrile, methanol and water were purchased from Burdick & Jackson, NJ. Mass spectrometry grade formic acid was purchased from Sigma- Aldrich, (St Louis, MO). Internal standards namely, [15N]2-Tryptophan, Glutamic acid –d5, Gibberellic acid, Jasmonic acid, Thymine-d4, and Zeatine, were purchased from Sigma- Aldrich, (St Louis, MO). Another internal standard, [15N] Anthranilic acid was purchased from Cambridge Isotope, (Tewksbury, MA). The calibration solution containing multiple calibrants in acetonitrile/trifluroacetic acid/water was purchased from Agilent Technologies, (Santa Clara, CA). The metabolomic analyses of all samples were executed using the protocol described previously [21 (link), 27 (link)–31 (link)]. The raw data (LC-MS output) was normalized using internal standards.

Metabolites were extracted using acetonitrile/methanol (75:25; v/v) containing deuterated internal standards (25 μM thymine-d₄ [Sigma-Aldrich], 10 μM inosine-¹⁵N₄ [Cambridge Isotope Laboratories], 10 μM citrulline-d₇ [Sigma-Aldrich], 25 μM phenylalanine-d₈ [Cambridge Isotope Laboratories], and 10 μM valine-d₈ [Sigma-Aldrich]). The samples were separated using a 2.1 × 100 mm 3.5-μm Xbridge amide column (Waters). Mobile phase A was 95:5 (v/v) water/acetonitrile, with 20 mM ammonium acetate and 20 mM ammonium hydroxide (pH 9.5). Mobile phase B was acetonitrile. For amide-negative mode, the chromatography system consisted of a 1260 Infinity autosampler (Agilent) connected to a 1290 Infinity HLPC binary pump system (Agilent). The eluents were detected in negative mode on a coupled 6490 QQQ mass spectrometry equipped with an electrospray ionization source. The settings were as follows: sheath gas temperature, 400 °C; sheath gas flow, 12 L/min; drying gas temperature, 290 °C; drying gas flow, 15 L/min; capillary, 4000 V; nozzle pressure, 30 psi; nozzle voltage, 500 V; and delta EMV, 200 V. Detailed methods have been described previously⁶² .

Reagents such as acetonitrile, methanol and water for high-performance liquid chromatography were purchased from Burdick and Jackson. Formic acid and internal standards, which included a mixture of Jasmonic acid, [¹⁵N]₂-Tryptophan, Thymine-d4, Glutamic acid-d5, Gibberellic acid, Trans-Zeatin, [¹⁵N]-Anthranilic acid and Testosterone-d3, were purchased from Sigma-Aldrich. For UDP-N-Acetyl glucosamine quantification, [¹³C]₆ N-acetyl glucosamine was used as an internal standard and the calibration curve for quantification was generated using the purified metabolite purchased from Sigma-Aldrich.