Q exactive plus hybrid quadrupole orbitrap mass analyser

Metabolites were extracted from frozen milled samples of each accession of the PMiGAP and were performed in four replicates of each accession. The seed samples (~50 mg ± 1 mg) were placed into 2 mL microcentrifuge tubes, each containing a single 5 mm diameter stainless-steel ball (acetone cleaned). Samples were immediately flash frozen in liquid N₂ and homogenized using a ball mill and put on ice to which 1 mL of extraction solution (chloroform/methanol/water, 1:2.5:1, v/v/v) was added followed by incubation at 4 °C for 15 min. The samples were further centrifuged at 14,000 rpm for 5 min at 4 °C and returned to the ice. The aqueous supernatant was transferred into new tubes for MS analysis. The extracted samples (100 µL) were transferred into the MS vials with inserts and analyzed for FIE-HRMS (flow infusion electrospray ionization high-resolution mass spectrometry). The four independent replicates were made for each sample. Metabolite fingerprinting was performed by FIE-HRMS using a Q Exactive Plus Hybrid Quadrupole Orbitrap Mass Analyser with an Acella ultra high-performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific, Bremen, Germany). The sample was injected into the capillary column in a randomized order. The m/z (mass-ion) values were generated in both positive and negative ionization modes as described by Skalska et al. [20 (link)].

Seedlings were harvested on the 7th day after germination and used for metabolite extraction. Each replicate represented a pool of fifteen seedlings with shoot and roots collected separately. The shoot and root samples (~ 7 mg ± 1 mg) of each tef accession (n = 3) were placed into 2 mL microcentrifuge tubes, each containing a single 5 mm diameter stainless-steel ball (acetone cleaned). Samples were immediately flash frozen in liquid N₂ and homogenized using a ball mill, to which 250 µL of ice-cold extraction buffer (chloroform/methanol/water, 1:2.5:1, by vol.) was added followed by incubation at 4 °C for 15 min. The samples were further centrifuged at 21,000×g for 5 min at 4 °C to separate the supernatant. Metabolite fingerprinting was performed for 100 µL of the extracted samples by flow infusion electrospray ionization high-resolution mass spectrometry (FIE-HRMS) using a Q Exactive Plus Hybrid Quadrupole Orbitrap Mass Analyser with an Acella ultra high-performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific, Bremen, Germany). The sample was injected into the capillary column in a randomized order and the m/z (mass-ion) features were generated in both positive and negative ionization mode. (The derived results are presented in Supplementary Tables S2 to S5).