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Metabolomics mammalian cells
==Work on ice during all the procedures!==
Prepare 4.2 N NH4OH: 100 μL of conc. NH4OH + 219 μL water.
Extraction buffer: Ethanol / water / diethyl ether / pyridine (15:15:5:1) + NH4OH (final concen: 2.1 mM)
For 12 samples, 3ml / 3ml / 1 ml / 0.2 ml / 3.6 μl 4.2 N NH4OH
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Step 1. Add 150 μL extraction buffer to the cell pellet (in 1.5 ml Eppendorf tube), pipette up and down to generate a homogenous suspension.
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Step 2. Add standard mixtures:
Component Each sample /μL (5 μL in total) C17 Sphingosine (Stock 1 mM; final conc. 0.04 nM) 0.04 C17 Sphinganine (Stock 1 mM; final conc. 0.04 nM) 0.04 C18 deoxy-Sphinganine (Stock 1 mM; final conc. 0.04 nM) 0.04 C17 Sphingosine-1P (Stock 2.5 mM; final conc. 4 nM) 0.16 C17 Sphinganine-1P (Stock 2.5 mM; final conc. 4 nM) 0.16 EtOH 4.56 -
Step 3. Vortex at 4 ºC for 10 min; Incubate on ice for 20 min
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Step 4. Centrifuge at max. speed for 2 min at 4 ºC
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Step 5. Transfer supernatant to a new 1.5 mL Eppendorf tube; add 150 μL new extraction buffer to the pellet.
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Step 6. Vortex at 4 ºC for 10 min; centrifuge at max. speed for 2 min at 4 ºC
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Step 7. Transfer supernatant to the tube used in Step 5; centrifuge the combined supernatant at max. speed for 2 min at 4 ºC
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Step 8. Transfer 150 μL supernatant to an amber glass ==with insert==; transfer the rest supernatant (~ 150 μl) to another amber glass with insert as backup.
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Step 9. Dry samples in speedvac (first 40 ºC, then 50 ºC)
Derivatization
Controls
Blank: 10 μl formic acid;
Amino acids: 5 μl serine + 5 μl Alanine;
Sphingoid bases: 2 nmol + 10 μl formic acid.
- Add 10 μl of formic acid to the samples
- Add 70 μl Borate buffer containing 15N13C-Valine
- Remove the bubble in the bottom of the tube
- Sonicate 5 min
- Add 20 μl of AQC (2.85 mg/ml in acetonitrile)
- Incubate 15 min at 55 ºC with 750 rpm of shaking
- Keep overnight at 24 ºC.