GC-MS Metabolome Analysis Protocol

GC-MS analysis was performed on the derivatized extracted metabolome according to Troisi et al. [15 (link)] with a few minor changes. Briefly, 2 µL of the sample solution was injected into the GC-MS system (GC-2010 Plus gas chromatograph coupled to a 2010 Plus single quadrupole mass spectrometer; Shimadzu Corp., Kyoto, Japan) equipped with a 30-m, 0.25-mm ID CP-Sil 8 CB fused silica capillary GC column with 1.00-µm film thickness from Agilent (Agilent, J&W Scientific, Folsom, CA, USA), using He as a carrier gas. The initial oven temperature of 100 °C was maintained for 1 min and then raised by 6 °C/min to 320 °C with a further 2.33 min of hold time. The gas flow was set to obtain a constant linear velocity of 39 cm/s, and injections were performed in the splitless mode. The mass spectrometer was operated in electron impact (70 eV) in the full-scan mode in the interval of 35–600 m/z with a scan velocity of 3333 amu/s and a solvent cut-off time of 4.5 min. The complete GC analysis duration was 40 min. Untargeted metabolites were identified by comparing the mass spectrum of each peak with the NIST library collection (NIST, Gaithersburg, MD, USA).