BAF_Protocol_009 Metabolomics: Lipid Extraction
Nicholas Sherman
Abstract
This protocol uses a similar method to Protocol_008 but retains the chloroform phase containing lipids and other very hydrophobic molecules. Extractions are done in a way to make sure the final sample is mostly lipids. This type of sample will need to be run on a different column using different buffers from those used for the aqueous/methanol metabolites.
Steps
Liquid Samples: Urine, Plasma, Culture Media
To each sample containing 100 uL add 750 µL of -20°C cold Chloroform:methanol (2:1) mixture and vortex
Shake tubes vigorously for 30 min at 4°C in temperature temperature-controlled thermal shaker
Add 400 µL of water, shake vigorously, and centrifuge for 10 min at 10000 rpm for phase separation
Discard the top aq. methanolic phase (if interested in soluble metabolite, recover this phase. See BAF_Protocol_008)
Recover the Lower phase to new clean Eppendorf
To each tube with 500 µL of chloroform phase, add 500 µL of cold Chloroform:methanol (2:1) mixture
Vortex and shaken vigorously for 30 min at 4°C in temperature controlled thermal shaker
Add 200 µL of water, shake vigorously, centrifuge for 10 min at 10000 rpm and recover the lower organic phase as a lipid mixture in glass tubes and store at -80°C
Before running, add 10 µL of Avanti Splash Lipidomix to each sample as internal standard and dry samples under gentle stream of N2 using a Recti-VapTM Evaporator (Thermo Fisher Scientific) at 40°C
Reconstitue in 110 µL of methanol:isoproponal (1:1)
Transfer 100 uL to borosilicate glass inserts kept inside a screw-capped glass autosampler vials (Agilent)
Solid samples: Cell Media, Stool, Tissue
Place the sample in reinforced tubes: Frozen tissue slice or lyophilized stools. For cell pellets – mix well with 50-100 uL of water transfer solution and suspended cells to reinforced tubes
To each sample add 5 stainless steel balls, 750 µL of -20°C cold Chloroform:methanol (2:1) mixture.
Disrupt cells/tissues with Fisher Bead Mill 24 (speed: 5m/s, time: 20 sec, number of cycle: 3, dwell/pause between runs: 10 sec).
Shake tubes vigorously for 30 min at 4°C in temperature temperature-controlled thermal shaker
Add 400 µL of water, shake vigorously, and centrifuge for 10 min at 10000 rpm for phase separation. Discard the top aq. methanolic phase (if interested in soluble metabolite, recover this phase. See BAF_Protocol_08)
Recover the Lower phase to new clean Eppendorf - extract lipids. Perform steps 06-11 from the above protocol
