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你想做代谢组，仪器条件一定要够好，此外，实验室里要有人工环境植物栽培箱，可以控制温度、湿度、光照、CO2浓度等。
你老板要有足够的经费，因为鉴定结构只能用GC-MS和LC-MS-MS，甚至用LC-NMR。
Metabolomic Analysis
Protocol for Plant Leaf Metabolite Profiling
(AG Fiehn, status 01.05.00)
Grow plants
Think about the requirements for obtaining statistical significance before you grow and harvest your plants!
Harvest plant leaves
Harvest 300 ± 30 mg FW tissue in 2 mL Eppendorf tubes at an appropriate day time. If using a phytotron, try to do the whole harvest in about 15-30 min. If not, local increases in CO2 concentration might lead to metabolic changes, so quickly weigh and freeze your tissue in liquid N2.
Alternatively, do rapid lyophilization of your tissue to obtain approximately 30 mg dry weight. If you have less than 300 mg FW, take 100 mg or 200 mg and appropriately alter the fractionation protocol. If you have less than 100 mg FW, alter both the fractionation and the derivatization protocols. If you have less than 10 mg FW, alter both protocols and alter the GC conditions (inject 3 µL out of 10 µL total volume in splitless mode). If you have less than 1 mg FW, alter all protocols and analyze by selected ion monitoring on m/z 73, 174, 204, 218, and 361.
Do not use tissue stored at -80°C for longer than four weeks as we have evidence that considerable metabolic changes occur after this time.
Extraction
Pre-chill Retsch ball-mill tube holders in liquid N2. Quickly place a ball in each of the sample tubes, put four samples into each of the ball-mill tube holders and homogenize the tissue for 1 min at 60% speed. Immediately put the tube holders and the samples back into liquid N2.
If you do not have a ball-mill, you may also grind the tissue in liquid N2 using a mortar and pestle, etc.
Take out the sample tubes one by one and immediately add 1.4 mL methanol (100%) in order to stop enzymatic activity. Vortex thoroughly. Add 50 µL of a ribitol stock solution (0.2 mg/mL H2O) and 50 µL of a nonadecanoic acid methyl ester stock solution (2 mg/mL CHCl3) as internal references. Add 50 µL water, vortex and check pH (should be pH 5-6). Shake at 70°C for 15 min (briefly open Eppendorf tubes after 1 min). Centrifuge at 14000 g for 3 min. Give supernatant into a 6-mL glass tube that is equipped with screw caps with teflonized inlays. Add 1.4 mL pure water (conductivity < 0.05 µS)), vortex, and check pH. Add 750 µL CHCl3 to the remaining centrifugate, vortex, and shake at 37°C for 5 min. Centrifuge at 14000 g for 3 min and transfer the supernatant into the MeOH/water solution of the glass vial. Vortex and centrifuge at 4000 rpm for 15 min.
Fractionation
Separate the upper (polar) phase into two new glass vials, one containing 1 mL and the other portion containing the rest (~1.8 mL). Take care not to carry over parts of the lipophilic fraction or insoluble residues. Close both new glass vials with parafilm, punch a little hole in the parafilm, and dry them in a speed vac concentrator overnight.
Derivatization
Lipid phase
The remaining lipid phase is treated as follows: Take out 100 µL for LC/MS analyses and refrigerate. To the remaining ~ 700 µL lipophilic phase, add 900 µL CHCl3. Add 1 mL MeOH containing 3% v/v H2SO4. Transmethylate lipids and free fatty acids for 4 h at 100°C.
Take care that your glass vial is sealed with a teflonized seal, and not with rubber. Otherwise, you will lose a lot of your solution and will find many rubber additives in your sample.
Extract your solution two times by adding 4 mL H2O, vortexing, centrifuging at 4000 rpm, and discarding the water phase. Dry the remaining chloroform phase over anhydrous Na2SO4 and transfer the supernatant into a new glass vial. Concentrate to about 80 µL. Add 10 µL pyridine and 10 µL MSTFA to the remaining 70 µL portion, silylate for 30 min at 37°C, and inject 2 µL into the GC/MS with a split ratio of 25:1.
Polar phase
Add 50 µL of methoxyamine hydrochloride (20 mg/mL pyridine) to the dried (1 mL) fraction of your polar phase. Incubate for 90 min at 30°C with continuous shaking. Add 80 µL of MSTFA for 30 min at 37°C and wait 120 min at 25°C before injection. Inject 2 µL, split 25:1. The second portion of the dry polar fraction can be used for LC/MS analyses or stored frozen at -80°C.
