Dionex ultimate 3000 rapid separation

Before the assay, protein samples were purified though size-exclusion chromatography and evaluated by SDS-PAGE. Then in-gel digestions were performed to prepare the mass spectrum samples (55 (link)). For molecular weight determination, Fur and Fur^YdiV were desalted and diluted to 1 mg/ml in 10 mM Tris–HCl pH 8.0 buffer without trypsin digestion.
HPLC–MS/MS was performed on the Dionex UltiMate 3000 Rapid Separation (RSLC) system (Thermo Scientific) coupled with an ESI-Q-TOF mass spectrometer (Bruker Daltonics). Proteins were separated on an XBridge Protein BEH C4 Column (2.1 mm × 50 mm I.D., particle size 3.5 μm) at 40°C with a mobile phase system of 0.1% formic acid (Sigma) in Milli-Q filtered water (A) and 0.1% formic acid (Sigma) in acetonitrile (Fisher Scientific) (B). The following gradient program was applied at a flow rate of 0.3 ml/min: 0–5 min, 95% A + 5% B; 5–30 min, 95–5% A + 5–95% B; 30–35 min, 5% A + 95% B; 35–50 min, 5–95% A + 95–5% B; and 50–55 min, 95% A + 5% B. The HPLC–MS/MS analysis was performed by using OTOF control software (Bruker Daltonics), and the protein molecules were calculated by charge deconvolution via Data Analysis software (Bruker Daltonics).

The surface metabolome was extracted in the laboratory by dipping each frond in 5 mL of LC-MS grade methanol (Carlo Erba, Peypin, France) during 5s according to [38 (link)] and subsequent sample preparation is detailed in SI. Analyses were performed on a UHPLC-ESI-HRMS system (Dionex Ultimate 3000 rapid Separation; ThermoFisher Scientific, Illkirch, France) with an analytical core-shell reversed phase column (150 × 2.1 mm, 1.7 μm, Kinetex Phenylhexyl; Phenomenex, Le Pecq, France) coupled with a QToF Impact II mass spectrometer (Bruker Daltonics, Bremen, Germany) in positive mode (details in SI).

A Dionex Ultimate 3000 Rapid Separation (RS) UPLC system (Thermo Fisher Scientific Inc., USA) with a Thermo Scientific Q Exactive Orbitrap Hybrid Tandem mass spectrometer and Heated-Electrospray Ionisation (H-ESI II) were used to identify anthocyanins profiles in dried MP using the method by Azman et al. [19 (link)] with slight modifications. Chromatography was carried out on a Purospher START RP18 end-capped column (5.0 µm, 4.6 mm i.d $\times$ 250 mm) temperature maintained at 30 °C. The mobile phases consisted of Solvent A (formic acid:water; 2:98, v/v) and Solvent B (methanol; 100, v/v) at a flow rate of 0.8 mL/min with 10 µL injection volume. The gradient elution program was used as followed: 0–19 min, 15% B; 19–38 min, 35% B; 38–50 min, 60% B; 50–56 min, 80% B; 56 min, 15% B. Mass spectrometry (MS) spectra were operated in the positive and negative ion mode between m/z 100 and 1500 at a scan resolution of 70,000 (full MS scan) and 35,000 (ddMS2 scan). The Qual Browser of the Xcalibur software (Thermo Scientific, USA) was used to analyze the acquired data for MS analysis.