Determination of incorporation of 34S into plant GSH and GSLs by LC-ESI-Q-ToF–MS

To determinate the ³⁴S incorporation into plant metabolites, ultra-high-performance liquid chromatography–electrospray ionization–high resolution mass spectrometry (UHPLC–ESI–HRMS) was performed with a Dionex Ultimate 3000 series UHPLC (Thermo Scientific, Germany) and a Bruker timsToF mass spectrometer (Bruker Daltonics, Bremen, Germany). UHPLC was done by applying a Zorbax Eclipse XDB-C18 column (100 mm × 2.1 mm, 1.8 µm, Agilent Technologies, Waldbronn, Germany) with a solvent system of 0.1% formic acid (A) and acetonitrile (B) at flow rate of 0.3 mL/min. The elution profile was as follows: 0 to 0.5 min, 5% B; 0.5 to 11.0 min, 5–60% B in A; 11.0 to 11.1 min, 60–100% B, 11.1 to 12.0 min, 100% B and 12.1 to 15.0 min 5% B. For the coupling of LC to MS, electrospray ionization (ESI) in positive and negative ionization mode, for GSH and GSL, respectively, was used. The mass spectrometer was set with the parameters: capillary voltage 4.5/3.5 kV, end plate offset of 500 V, nebulizer pressure 2.8 bar, nitrogen at 280 °C at flow rate of 8 L/min as drying gas. Acquisition was conducted at 12 Hz with a mass range from m/z 50 to 1500. At the beginning of each chromatographic analysis, 10 µL of a sodium formate-isopropanol solution (10 mM solution of NaOH in 50/50 (v/v%) isopropanol- water containing 0.2% formic acid) was injected into the dead volume for recalibration of the mass spectrometer with the expected cluster ion m/z values. Peak areas were integrated from extracted ion chromatogram traces of the monoisotopic molecular ion peak ([M + H]⁺, [M − H]⁻) and of the isotopologues that could be detected with an isolation width of m/z ± 0.002. Details of m/z values of isotopologues are listed in Table ^S2. First we calculated the percentage of single isotopologues (% isotopologue) as a proportion of the sum of all isotopologues for each single compound (i.e. % of the monoisotopic molecular ion peak = peak area of the monoisotopic molecular ion peak × 100%/(peak area of the monoisotopic molecular ion peak + (peak area of “isotopologue + 1”) + (peak area of “isotopologue + 2”) + (peak area of “isotopologue + 3”) + (peak area of “isotopologue + 4”). In order to determine the incorporation of ³⁴S, the ³⁴S/³²S ratio was calculated (³⁴S/³²S ratio = % “isotopologue + 2”/% of the monoisotopic molecular ion peak).