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boiling-water-protocol

Extraction of membrane glycerolipids from plant leaves or pellets of bacerial or fungal cells with boiling water for mass spectrometry

This protocoll works well for plant leaves, flowers, and soft roots (e.g. Arabidopsis)
For thick roots (Lotus, Medicago, etc)  use acidic chloroform-methanol extraction.
from bacteria/yeast pellets etc.

Use glass tubes with screw caps and teflon septa to avoid contamination of the lipid extract. Never use rubber septa in the caps. It is best to use fresh leaves directly taken from plant, not frozen leaves.

1._Cut material (leaf, flower, ca. 200 mg fresh weight, similar amounts from each sample) fresh from plant
   (or take bacterial cell pellet). Usually it is best to skip fresh weight measurements and determine dry weight later
   (see point 6).

2. Put material into glass tube (wide opening, can also use small beaker or flask covered with aluminum foil) with boiling
    water (Millipore water) (10-20 min) standing in 100°C water bath. This destroys all lipase activity. If you use a tube with
    screw cap, make sure that it has a teflon septum.

3. Remove water completely with a pipette.

4. Extract leaf with 1 vol CHCl3:MeOH 1:2 (v/v), harvest all (!) organic lipid extract (no phase separation).

5. Extract leaf with 1 vol CHCl3:MeOH 2:1 (v/v), harvest all (!) lipid extract (no phase separation). Make sure that leaf is white
    (not green) after second extraction. Otherwise repeat this extraction step. Collect the combined organic extracts in a
    separate tube. Tubes (e.g. culture tubes, 8 ml vol., Schott, # 233 175 11 59, with extra teflon septum/caps, Schott # 29
    240 08 06; replacement teflon discs # 29 248 08 05). Any other glass tubes with teflon septum/cap will also work fine.
    Can store the lipid extract at 4°C.

6. Determine the dry weight (after lipid extraction) of the leaf (should be around 10 mg). To this end, put the leaf residue
    (in glass vial) into oven at > 100 °C overnight. Next morning, determine dry weight of samples.

7. At this point, you can send samples (upright, in glass tubes with screw caps with teflon septa) at room temperature by
    normal mail for lipid quantification. You can also blow off the liquid with N2 gas. Then close the tube with cap and ship
    the dry lipid samples to us. In Bonn, we will continue like this:

8. To the combined chloroform fractions, add 1 vol chloroform, 1 vol methanol and 0.75 vol 300 mM NH4OAc solution, such
    that the final ratio of CHCl3:MeOH: NH4OAc-solution is: 2:1:0.75 (v/v).

9. Vortex, centrifuge, harvest lower (lipid) phase; take out all (!) of the chloroform phase, but avoid taking the aqueous
    phase. To the residual aqueous phase, add another 200 µl CHCl3/MeOH (2:1, v/v), vortex, centrifuge, harvest organic
    (lower) phase and combine with the previous chloroform phase.

10. Evaporated samples are dissolved in Q-TOF solvent for LC-MS.

11. Prepare an exact dilution (e.g. 1:10) in Q-TOF solvent and add internal standard mix.

Reference:
Roughan P, Slack C, Holland R (1978) Generation of phospholipid artefacts during extraction of developing soybean seeds with methanolic solvents. Lipids 13: 497-503

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