## 2 Materials and Methods
### Analytical Methods
#### 2.4.3 Non-Volatile Metabolome Analysis  
Ultra-high-resolution mass spectra were acquired using an FT-ICR-MS (SolariX, Bruker Daltonik, Bremen, Germany) equipped with a 12 Tesla superconducting magnet (Magnex Scientific Inc., Kidlington, UK) and an Apollo II electrospray ionization source (Bruker Daltonik, Bremen, Germany) operated in negative ionization mode. Samples were collected at the end of alcoholic fermentation and diluted at 5:100 \(v/v\) in pure methanol (LC-MS grade, Fluka, Germany). Quality controls (QC) were prepared by pooling equal amountsof all samples. QCs were analyzed at the beginning and end of the run and every ten samples to validate the repeatability of the measurement. The diluted samples and QC samples were injected at a flow rate of 120 uL/h into the electrospray ion source. Spectra were acquired with a time domain of 4 megawords within a mass range of m/z 147-2000. At total of 300 scans was accumulated for each sample. All the samples were injected randomly in the same batch to avoid batch variability. A resolving power of 400,000 at 300 m/z was achieved. All the spectra were calibrated externally on a methanol solution of arginine clusters (10 mg/L), and accuracy reached values of less than 0.1 ppm in day-to-day measurements. Additionally, internal calibrations were performed on a list composed by recurrent compounds in wine to keep only m/z peaks with a signal-to-noise (S/N) ratio of 4. Matrix Generator software (v. 0.4, Helmholtz-Zentrum Muenchen) was used to align each peak with a mass-accuracy window of 1 ppm. The quality of the analysis was controlled before any data processing (Supplementary Figure S1). Features were annotated using the in-house software NetCalc 2015 (v.1.1a, Helmholtz-Zentrum Muenchen). Finally, van Krevelen diagrams were generated by an Excel file according to the H/C versus O/C ratio of annotated metabolites. Annotation levels were in line with [PERSON] et al., 2017 [25]. Perseus 1.5.1.6 (Max Planck Institute of Biochemistry, Germany) was used to perform principal-component analysis (PCA), hierarchical cluster analysis (HCA), and analysis of variance (ANOVA). For HCA, the Euclidean distance and average linkage were chosen, and a threshold \(p\)-value of 0.05 was chosen for ANOVA. Multidimensional Stoichiometric Compounds Classification (MSCC) have been used to elucidate extracted biomarkers categories commonly defined as lipids, peptides, amino sugars, carbohydrates and polyphenols derivatives compounds [26]. Metabolites were identified by ultra-high-performance liquid chromatography (Dionex Ultimate 3000, ThermoFischer, Waltham, MA, USA) coupled to a MaXis plus MQ ESI-Q-ToF mass spectrometer (Bruker, Bremen, Germany). A reversed-phase liquid-chromatography (RP-LC) separation method was applied by injecting 5 uL in an Acquity UPLC BEH C18 1.7 um column 100 \(\times\) 2.1 mm (Waters, Guyancourt, France) to separate metabolites according to their polarity. Buffer A (acetonitrile 5% (\(v/v\)) with 0.1% (\(v/v\)) formic acid) and buffer B (acetonitrile with 0.1% (\(v/v\)) formic acid) were employed to elute metabolites. Detection was implemented in negative ionization mode with the following parameters: an electrospray (Nebulizer pressure = 2 bars and nitrogen dry gas flow = 10 L/min), ion transfer (end plate offset at 500 V) capillary voltage (at 4500 V), and acquisition (100-1500 m/z mass range). A mix of standard peptides and polyphenols was used for the UHPLC-Q-ToF-MS quality control. Experimental quality control (mix of samples) was used to guarantee system repeatability. All the samples were injected randomly in the same batch to avoid batch-to-batch variability. Calibration was processed with 1/4 diluted ESI Tuning Mix (v. 4.3, Bruker Daltonik GmbH). Features (couple of m/z-values and retention times) were fragmented using the AutoMS/MS function on the most intense features, with a frequency of 2 Hz. Fragmentation was done at three different collision energies: 15, 25, and 35 eV.