| MS Description
|
BioSource amount<br />
We weighed 70 … BioSource amount<br />
We weighed 70 mg dry weight (DW) of the lyophilized samples for CE-TOF-MS analysis, 5 mg DW for GC-TOF-MS analysis, 50 mg DW for LC-q-TOF-MS analysis to detect polar metabolites, and 15 mg DW for lipid profiling. <br />
<br />
Extraction for LC-q-TOF-MS to detect polar metabolites<br />
Fifty-mg DW of each sample was extracted in 50 volumes of extraction medium (methanol/water [2:5 v/v]) containing two reference compounds (0.5 mg/l flavonol-2’-sulfonic acid and1.0 mg/l ampicilin) using a mixer mill MM301 (Retsch) at a frequency of 20 Hz for 5 min at 4°C. After centrifugation for 10 min at 15,000 × g, the supernatant was transferred into a 2 ml tube. Thirty volumes of methanol were added to the tube and then extracted again using the mixer mill at a frequency of 20 Hz for 5 min at 4°C. After centrifugation for 10 min at 15,000 x g, the resulting supernatant was transferred into the tube. One hundred twenty-μl aliquot of the extracts was filtered using an Oasis® HLB μelusion plate (30 μm, Waters Co., Massachusetts, US). The extracts (100 μl) were transferred into a 2 ml tube and were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo Fisher Scientific). The extracts were dissolved by 100 μl of 20% aqueous methanol containing 0.5 mg l−1 lidocaine and 10-camphorsulfonic acid. <br />
<br />
LC-q-TOF-MS conditions to detect polar metabolites<br />
After preparation of the extracts, the sample extracts (1 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). The analytical conditions were as follows. LC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 μm, length 2.1 × 100 mm, Waters); solvent system, solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid); gradient program, 0.5% of solvent B at 0 min, 0.5% of solvent B at 0.1 min, 99.5% of solvent B at 12.0 min, 99.5% of solvent A and 0.5%B at 12.0 min, 0.5% of solvent B at 12.1 min, and 0.5% of solvent B at 15.0 min; flow rate, 0.3 ml/min; temperature, 40°C; MS detection: capillary voltage, +3.0 keV, cone voltage, 25.0 V, source temperature, 120°C, desolvation temperature, 450°C, cone gas flow, 50 l per h; desolvation gas flow, 800 l per h; collision energy, 6 V; mass range, m/z 100‒1500; scan duration, 0.1 sec; interscan delay, 0.014 sec; mode, centroid; polarity, positive; Lockspray (Leucine enkephalin): scan duration, 1.0 sec; interscan delay, 0.1 sec. The data were recorded using MassLynx version 4.1 software (Waters).<br /> version 4.1 software (Waters).<br />
|
