BAF_Protocol_008 Metabolomics: Soluble Metabolite Extraction
Nicholas Sherman
Abstract
This is a standard metabolite sample prep using chloroform/methanol and keeping the soluble (aqueous) phase. This method will produce a clean sample free of most lipids and very hydrophobic molecules. The samples are ready for MS analysis or storage dry at -80C.
Steps
Liquid Samples: Urine, Plasma, Cell 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.
Recover the top aq. methanolic phase as metabolite mixture and transfer to Eppendorf tube.
Dry the soluble metabolite samples under vacuum for 3-4 h. Store at -80°C until ready for LC-MS(/MS) analysis.
*Note: save the lower phase if you need to extract lipids. Go for BAF_Protocol_009.
Before running, reconstitute samples in 100 µL of 0.1% Formic acid in water containing 100X diluted Metabolomics QReSS heavy labeled standards. (https://www.isotope.com/userfiles/files/assetLibrary/MET_RSCH_QReSS.pdf)
Prepare a QC sample with 10 µL of each sample and transfer 50 uL of each sample to autosampler vials
Injection volume of each sample: 10 µL
Samples that need disruption/lysis: Tissue, Stools, Cell pellets
Place the sample into 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)
Follow steps #2 to #9.
