Determination of incorporation of 34S into plant metabolites by LC-ESI-Q-ToF-MS
For the determination of 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) and a Bruker timsToF mass spectrometer (Bruker Daltonics, Bremen, Germany). UHPLC was used 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 a flow rate of 0.3 mL/min. The elution profile was the following: 0 to 0.5 min, 5% B; 0.5 to 11.0 min, 5% to 60% B in A; 11.0 to 11.1 min, 60% to 100% B, 11.1 to 12.0 min, 100% B and 12.1 to 15.0 min 5% B. Electrospray ionization (ESI) in positive ionization mode (for GSH) and in negative ionization mode (for GSL) was used for the coupling of LC to MS. The mass spectrometer parameters were set as follows: capillary voltage 4.5/3.5 KV, end plate offset of 500 V, nebulizer pressure 2.8 bar, nitrogen at 280 °C at a flow rate of 8 L/min as drying gas. Acquisition was achieved 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 of the sample injection for recalibration of the mass spectrometer using 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. For details of m/z values of isotopologues see 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, the34S/32S ratio was calculated (34S/32S ratio = % “isotopologue + 2”/% of the monoisotopic molecular ion peak).