Glucosinolate Extraction and Desulfation

GSLs were extracted as previously reported [31 (link),32 (link)]. The dried plant parts of Rab sample (aerial part) and Split sample (flower, leaf, stem, siliquae, root) were ground to a fine powder, from which 100 mg were extracted for 5 min at 80 °C in 2 × 1 mL MeOH/H₂O (70:30 v/v) to inactivate the endogenous myrosinase. Each extract (1 mL) was loaded onto a mini-column filled with 0.5 mL of DEAE-Sephadex A-25 anion-exchange resin (GE Healthcare, Chicago, IL, USA) conditioned with 25 mM acetate buffer (pH 5.6). After washing the column with 70% MeOH and 1 mL of ultrapure water, optimal conditions for desulfation were set by adding buffer solution. Each mini-column was loaded with 20 μL (0.35 U/mL) of purified sulfatase and left to stand 18h at room temperature. The desulfo-GSLs were then eluted with 1.5 mL of ultra-pure H₂O, lyophilized and diluted to the 1 mL. The samples were stored at –20 °C until further analysis by UHPLC-DAD-MS/MS.