Methods for generating the MS files in the dataset <Glycyrrhiza Lepidota Leaf Mass Spectrometry Data>

LC-MS Data Acquisition:
Metabolic fingerprints were generated using C18-reversed-phase ultra-performance liquid chromatography-electrospray ionization-hybrid quadrupole-orbitrap mass spectrometer (Ultimate® 3000 HPLC, Q Exactive™, Thermo Scientific).  Chromatographic separation was accomplished on a reversed-phase C18 HSS T3 1.8 µm particle size, 2.1x100 mm column (Waters) with column temperature at 40 °C, solvent flow rate 0.45 mL/min, and injection volume 1 µL. A 20-minute gradient using mobile phases A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile was run according to the following gradient elution profile: initial, 15% B; 1 min, 15% B; 2 min, 50% B; 15 min, 98% B; 16 min, 98% B; 16.5 min, 15% B; 20 min, 15% B. The following MS conditions were used: full scan mass scan range: 130-1000 m/z, resolution: 35,000, data type: profile, desolvation temperature 350 °C, capillary voltage: 3800 V (+), 3300 V (-).  Xcalibur™ software version 2.1 (Thermo Scientific) was used to record the chromatograms and spectra. Metlin (Smith, Colin A., et al. "METLIN: a metabolite mass spectral database." Therapeutic drug monitoring 27.6 (2005): 747-751.) and Human metabolome database (Wishart, David S., et al. "HMDB 3.0—the human metabolome database in 2013." Nucleic acids research (2012): gks1065.) provided reference or in silico tandem mass spectra. 



Leaf Spray MS, we have implemented a mass spectrometry based metabolomics approach to sample metabolites from intact live plant tissue. This method has been successfully adapted from methods developed by Liu and coworkers (Liu, Jiangjiang, et al. "Leaf spray: direct chemical analysis of plant material and living plants by mass spectrometry." Analytical chemistry 83.20 (2011): 7608-7613.). Aerial parts of G. lepidota plants were harvested and leaves were selected for immediate analysis by leaf spray. An alligator clip was attached at the leaf base with the apex aimed at the MS inlet. Methanol (10 µL) was applied with a pipette twice during the one min. MS acquisition with full scan mass scan range 130-1000 m/z, polarity switching, and 70,000 resolution. A second leaf was used for fragmentation of the 30 most abundant ions (Table S1) using a normalized collision energy of 50 and a resolution of 17,500. A nanoelectrospray source was modified to administer 4.5 kV to a leaf via the alligator clamp and capillary temperature was set at 250 ˚C.
 
For Bulk Extraction MS, aerial parts were collected and dried in a 30 °C oven for three days. The dry material was ground in a Thomas Wiley laboratory mill model 4 (Thomas Scientific, Swedesboro, New Jersey, USA) with a 6 mm screen. Plant material (250 mg) was extracted for four hours with 1.5 mL of aqueous ethanol (ethanol: water, 70:30 v/v) and agitated at 700 RPM with a 2010 Geno/Grinder® (SPEX Sample Prep, Metuchen, NJ, USA). Extracts were centrifuged at 11,750g for 5 min. The supernatant was diluted 1:10 with 70% ethanol prior to LC-MS analysis.
 
For Leaf Dip Extraction MS, G. lepidota leaves of similar size were harvested by cutting the petioles at the stem. Each leaf was separately dipped in 1 mL methanol for 2 min and solvent was evaporated to dryness in a vacuum centrifuge (Speedvac). Residue was re-suspended in 50 µL of 80% acetonitrile:water (v/v) followed by vigerous mixing for 2 min. (Vortex mixer). Extracts were subjected to centrifugation at 2,600g for 10 min. and the resulting supernatants were used for LC-MS analysis. This method has been used by other researchers to produce extracts enriched for trichome metabolites for tomato metabolic profile analysis (Ghosh, Banibrata, Thomas C. Westbrook, and A. Daniel Jones. "Comparative structural profiling of trichome specialized metabolites in tomato (Solanumlycopersicum) and S. habrochaites: acylsugar profiles revealed by UHPLC/MS and NMR." Metabolomics 10.3 (2014): 0.).