Quantifying Glucosinolate Hydrolysis by LC-MS/MS

Assays consisted of 50 µL crude beetle protein extracts or recombinant protein and 50 µL glucosinolate solution containing a mixture of 2-hydroxy-3-butenyl-, 3-butenyl-, 4-methylsulfinylbutyl-, 4-methylthiobutyl-, indol-3-ylmethyl-, benzyl-, 2-phenylethyl- glucosinolate, and sinalbin, each at 2 mM, in 20 mM MES buffer (pH 5.2). After incubation at 35 °C for 2 h, assays were stopped by adding 200 µL of pure methanol and 200 µL of 10% (w/v) aqueous DEAE-Sephadex A-25 (GE Healthcare Life Science, Pittsburgh, PA, USA) suspension to remove remaining glucosinolates. Samples were filtrated through a syringe filter (0.2 μm, Pall Corporation, Dreieich, Germany) into glass vials and stored at −20 °C until analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Assays performed with boiled protein samples served as a background control. Desulfo-glucosinolates were quantified by LC-MS/MS using an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, USA) connected to an API3200 tandem mass spectrometer (AB Sciex Germany GmbH, Darmstadt, Germany) as described in Beran et al.^{15 (link)}. Multiple-reaction monitoring (MRM) was used to monitor analyte parent ion-to-product ion formation (Table S4). Data analysis was performed using Analyst Software 1.6 Build 3773 (AB Sciex). Desulfo-glucosinolates were quantified via external calibration curves.