GC-MS Analysis

Before derivatization, the samples were transferred to a CentriVap to be dried at 40°C for 15 min. Samples were derivatized as previously described (Kind et al., 2009; Vermeersch et al., 2014). 10 μL of 20 mg/mL O-methylhydroxylamine hydrochloride (MP Biomedicals, LLC, Santa Ana, CA, United States) in pyridine was added to each dried sample and shaken at 1400 rpm for 90 min at 30°C. 90 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) + 1% trimethylchlorosilane (TMCS) (Thermo Scientific, Lafayette, CO, United States) was then added to the samples and shaken at 1400 rpm for 30 min at 37°C. Samples were centrifuged at 21,100 g for 3 min, and 50 μL of the supernatant was added to an autosampler vial. Samples were spiked with 0.25 μL of a retention time standard solution composed of fatty acid methyl esters (FAMES) and an internal standard of nonadecanoic acid methyl ester dissolved in dimethylformamide. In parallel, a quality control (QC) sample was prepared by removing 150 μL of extract from each sample and aliquoting 1.15 mL for experiments comparing the metabolic changes based on lycopene production levels. For experiments comparing the effect of the mevalonate pathway, 75 μL was removed. 1.15 mL and 0.65 mL of QC was aliquoted for each experiment respectively, dried, and derivatized with each batch of 9–10 samples. At the beginning of the GC-MS run, the QCs were injected once and repeated again after every 4–5 sample injections to allow for downstream correction for batch effects. A derivatization blank was prepared and run with every batch of samples.

A LECO Pegasus 4D instrument with an Agilent 7683B autosampler, Agilent 7890A gas chromatograph and time-of-flight mass spectrometer (TOF-MS) was used to analyze the samples. The first column was an HP-5, 28 m long × 0.320 mm ID × 0.25 μm film thickness (Agilent, Santa Clara, CA, United States), and the second was an Rtx-200, 1.75 m long × 0.25 mm ID × 0.25 μm film thickness (Restek, Bellefonte, PA, United States). More detailed gas chromatography, autosampler, and mass spectrometry methods are provided in the Supplementary Material.