Aoc 5000 plus with gcms tq8040

A total of 100 etiolated seedlings at 3 days after induction of seed germination (DAI) were collected in a tube in liquid nitrogen and lyophilized. The samples were extracted with a bead shocker in a 2 ml tube with 5 mm zirconia beads and 80% MeOH for 2 min at 1000 rpm (Shake Master NEO; Biomedical Sciences). The extracted solutions were centrifuged at 104 g for 1 min, and 150-μl centrifuged solution and 10-μl 2 mg/L (UL-¹³C₆^glc)-Suc (Omicron Biochemicals, South Bend, IN, United States) were dispensed into a 1.5-ml tube. After the solution was dried with a centrifuge evaporator (Speed vac; Thermo), 100-μl Mox reagent (2% methoxyamine in pyridine; Thermo) was added to the 1.5-ml tube, and the metabolites were methoxylated at 30°C and 1,200 rpm for approximately 6 h with a thermo-shaker (BSR-MSC100; Biomedical Sciences). After methoxylation, 50-μl 1% v/v trimethylchlorosilane (TMS; Thermo) was added to the 1.5-ml tube. For TMS derivatization, the mixture was incubated for 30 min at 1,200 rpm at 37°C as above. Finally, 50 μl of the derivatized samples were dispensed into vials for GC-QqQ-MS analysis (AOC-5000 Plus with GCMS-TQ8040; Shimadzu) as described elsewhere (Tabeta et al., 2021 (link)). The raw data were collected, and GC-MS peak areas were calculated with GCMS software (Shimadzu). Suc content was quantified per etiolated seedling based on an internal standard.

For GC-MS/MS analysis, 100 μl aliquots of 250 μM πMH and τMH solutions were dispensed into separate 1.5 ml tubes. After evaporation of the solution using a centrifuge evaporator (SpeedVac, Thermo Fisher Scientific) and addition of 100 μl Mox regent (2% methoxyamine in pyridine, Thermo) to each tube, the metabolites were methoxylated at 30°C for approximately 6 h with shaking at 1,200 rpm using a thermo shaker (BSR-MSC100, Biomedical Sciences). Next, 50 μl of 1% (v/v) trimethylchlorosilane (TMS, Thermo Fisher Scientific) was added, and TMS derivatization was carried out by incubating the mixture at 37°C for 30 min with shaking at 1,200 rpm. Finally, 50-μl aliquots of the derivatized samples were dispensed into vials for GC-MS/MS analysis (AOC-5000 Plus with GCMS-TQ8040, Shimadzu). πMH-2TMS and τMH-2TMS were annotated using total ion chromatographs obtained by GC-MS/MS in scan mode, and MRM transitions (parent ion > daughter ion) were determined to be 218.00 > 73.10 for πMH-2TMS and 196.00 > 73.10 for τMH-2TMS. Plant extracts were derivatized in the same manner as used for πMH and τMH, and MHs in the extracts were analyzed by GC-MS/MS in MRM mode. Raw data collection was performed using GCMS Solution software (Shimadzu). GC-MS/MS parameters have been detailed previously (Tabeta et al., 2021 (link)).