Q exactive quadrupole orbitrap mass spectrometry system

Crocetin dialdehyde was dissolved in 100 μL of incubation buffer (200 mm pyrophosphate, 200 mm NaCl, pH 7.5, 2 μL of 100 mm NAD⁺), and 80 μL of crude protein and water were added to give a total volume of 200 μL. Incubations were performed at 28 °C for 30 min, stopped by addition of 1 mL of acetone, extracted with light petroleum/diethyl ether (1:4, v/v). The reactions were analyzed by LC-APCI(+)-MS, using a Q-exactive quadrupole Orbitrap mass spectrometry system (ThermoFisher Scientific, Madrid, Spain), coupled to a HPLC system equipped with a photodiode array detector (Dionex, Madrid, Spain) as described [9 (link)].

ABA and ABA catabolites detection and quantification were performed as reported in Diretto et al. (2020) (link) and Barja et al. (2021) (link). Briefly, 50 mg freeze-dried grounded cane samples were extracted using unbuffered Tris-ethyl acetate as reported before (Welsch et al., 2008 (link)). LC-HRMS was carried out using an Ultimate UHPLC system with a photodiode array detector (Dionex), and a Q-exactive quadrupole Orbitrap mass spectrometry system (Thermo Fisher Scientific; LC-HRMS) equipped with an electrospray ionization (HESI) source, operating in negative ion mode, as previously described (Di Meo et al., 2019 (link)) with the following modifications: with nitrogen as sheath and auxiliary gas set at 35 and 25 units, respectively. The vaporizer and capillary temperatures were set at 280 and 320°C, respectively. The discharge current was set to 4.0 μA and S-lens RF level set at 50. Internal standard-based quantification was carried out using the MS data, while retention times and MS2 fragmentation patterns were used for ABA identification by using authentic reference standards (trans-ABA from OlChemIm and ()-ABA from Sigma, St. Louis, MO, United States).

Determination of ABA, its glycosylated form (ABA-GE), and catabolites (phaseic and dihydrophaseic acid) in flavedo, pulp, bark and xylem of the different genotypes were performed as described by Diretto et al. (2020) (link) with slight modifications. Briefly, frozen tissues were lyophilized and ground to a fine powder, and replicate samples of 200 mg of each genotype were extracted as previously described (Welsch et al., 2008 (link)). LC-HRMS was carried out using a Ultimate UHPLC-DAD (Dionex) coupled to a Q-Exactive quadrupole Orbitrap mass spectrometry System (Thermo Fisher Scientific), equipped with a C18 Luna column (150 x 2.0mm, 3μm) as described before (Noronha et al., 2022 (link)). Internal standard-based quantification was carried out using the MS data and the quantification software available in the Xcalibur 7.0 software package (Thermo Fisher Scientific, Bremen, Germany). Results are expressed as fold changes with respect to the internal standard.