FT-ICR-MS based untargeted metabolomics of Melilotus officinalis plants grown in different soil treatments

This study reports the application of the leguminosae <em>Melilotus officinalis</em> in soil recovery and as a source of bioactive compounds. Plants were cultivated in semiarid soil under four different fertilizer treatments: urban waste compost at 10 t/ha (C-Low) and 20 t/ha (C-High), inorganic fertilizer (Inorg) and a Control (without any fertilizer). Metabolite composition of plants' leaves was analyzed through Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Metabolite extraction was performed from frozen lyophilized leaf samples (eight biological replicates per treatment) with 1 mL of 50% methanol/water (LC–MS grade, Merck). Then, the methanolic sample extract was diluted 1000-fold in methanol and analysed by direct infusion on a 7-Tesla SolariX XR Fourier Transform Ion Cyclotron Resonance Mass Spectrometer, equipped with a ParaCell (FT-ICR-MS, Brüker Daltonics). Leucine enkephalin (YGGFL, Sigma Aldrich) was added as internal standard ([M+H]+ =556.276575 <em>m/z</em>), at a concentration of 0.1 µg/mL, and formic acid (Sigma Aldrich, MS grade) was added at a final concentration of 0.1% (v/v) to all samples. Spectra were acquired in positive electrospray ionization (ESI+), in magnitude mode, with an acquisition size of 4M, and recorded between 100 and 1500 <em>m/z</em>. The accumulation time was 0.1 seconds, and 100 transients were accumulated for each spectrum, zero-filled and apodised (half-sine). Online calibration was performed using the monoisotopic <em>m/z</em> value of leucine enkephalin. The software Bruker Compass data Analysis 5.0 (Brüker Daltonics, Bremen, Germany) was used to process and retrieve the mass lists, considering peaks with a minimum signal-to-noise ratio of 4. The alignment of the raw data from the different replicas for each spectrum dataset was performed with Metaboscape 5.0 (Brüker Daltonics, Bremen, Germany) using T-ReX 2D (MRMS single spectrum) algorithm, with the intensities normalized to the sum of all intensities in each sample. M/z values that appeared only in one analysed sample were removed from the table.