Considerations of Sample Preparation for Metabolomics Investigation

Abstract

Sample preparation is the gateway to metabolomic analysis, the importance of which cannot be overemphasized. There are general rules of thumb for sample preparation that help maximize sample integrity and metabolite recovery. The wide range of variations in metabolite functional groups, polarity, sizes, and stability precludes the use of a single extraction method in metabolomic studies. Common extraction methods for polar metabolites that utilize trichloroacetic acid or aqueous acetonitrile are suitable for both nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis while others that use chloroform/methanol/water partition or boiling water may not. Control of extract pH is crucial for consistent NMR assignments and chemical derivatization-linked MS analysis. Sequential polar and lipid extractions reduce sample size requirement and provide a better coverage for direct-infusion MS analysis of lipids, possibly by removing interfering salts. Cleanup of sample extracts, such as removal of fine particles or interfering cations, is often necessary but should be limited to reduce loss of metabolites.

Appendices

Appendices

1.1 Protocol for Preparing Adherent Mammalian Cells for Metabolite Extraction

1.1.1 Trypsinization Method

• 1. When feasible, keep cells ice cold during all processing steps prior to freezing.

• 2. Harvest cells by trypsinization and centrifugation at 280 × g for 5 min at 4°C.

• 3. Wash cell pellet by resuspension in excess ice-cold phosphate-buffered saline (PBS) (e.g., 10–15 mL per 10⁷ cells), followed by centrifugation at 280 × g for 5 min at 4°C.

• 4. Resuspend cell pellet in ice-cold PBS (e.g., 1 mL per 10⁷ cells) and transfer cell suspension to a pre-tared microfuge tube.

• 5. Centrifuge cell suspension at 1,700 × g (or at maximal g force without disrupting cell integrity) for 5 min at 4°C so that as much PBS as possible can be removed by pipetting with a fine-tip transfer pipette.

Note: PBS contributes salts to the sample, which can be problematic for both MS and NMR analyses.

• 6. Obtain wet weight of cell pellet before flash freezing in liquid N₂.

• 7. Store frozen cell pellet at −80°C or lyophilize and obtain dry weight before storage.

1.1.2 Solvent-Quenching Method

• 1. Place cell culture plate on ice, remove culture medium by vacuum suction, and wash cells three times with ice-cold PBS by vacuum suction.

• 2. In the last wash, slant plate to drain PBS and remove as much PBS as possible by vacuum suction. Keep plate on ice for all subsequent procedures.

Note: This is important to minimize salt contribution.

• 3. Add cold methanol (kept at −80°C) or acetonitrile (kept at −20°C) to drained plate to quench cell metabolism. Adjust solvent volume so that the plate surface is covered fully with solvent, e.g., 1 mL for 10-cm-diameter plate or 2.5 mL for 15-cm-diameter plate.

• 4. Scrape plate with a cell scraper to collect cell mass and transfer cell mass to a 15-ml conical polypropylene (PP) centrifuge tube.

• 5. Add another equal volume of cold methanol or acetonitrile to plate; scrap and collect cells as in step 4.

• 6. Add nanopure water to plate in a ratio of water:methanol (1:1) or water:acetonitrile (1.5:2); scrap and collect cells as in step 4.

• 7. Add chloroform to the cell homogenate in a ratio of CHCl₃:water:methanol (1:2:2) or CHCl₃:water:CH₃CN (1:1.5:2).

Note: All solvents used should be of optima or HPLC grade.

• 8. Mix aqueous and CHCl₃ layers rigorously in the 15-mL tube before centrifugation at 3,000xg for 20 min at 4°C to separate the layers.

• 9. Transfer the upper aqueous and lower chloroform layers to 1.5-mL PP microfuge tube and 1.5-mL screw-cap glass vials, respectively, using a fine-tip transfer pipet for aqueous layer and PP gel-loading tip for chloroform layer.

Note: Avoid pipetting the protein/cell debris in between the aqueous and chloroform layers.

• 10. Dry aqueous and chloroform extracts in a vacuum centrifuge.

1.2 Protocol for Preparing Blood Plasma for Metabolite Extraction

• 1. Collect blood into purple-top vacutainer containing K₃-EDTA as anticoagulant. Care should be taken to avoid lysis of the blood.

Note: EDTA is compatible with both MS and NMR analyses; for NMR, it helps remove the influence of interfering cations, such as paramagnetic Fe³⁺ and Cu²⁺.

• 2. Immediately keep blood sample on ice during transport.

• 3. Centrifuge blood (within 30 min–1 h of collection) at 3,939 × g for 15 min at 4°C.

• 4. Aliquot plasma into separate microfuge tubes and flash freeze in liquid N₂.

• 5. Store plasma samples at −80°C until analysis.

1.3 Protocol for Freeze Clamping

• 1. Immerse an aluminum clamping device (cf. Fig. 6) in liquid N2 bath to preequilibrate.

  Fig. 6.

  

  Freeze clamp.

• 2. Place tissue(s) in the middle of the frozen clamp and quickly close the handles to flatten the sample(s), immediately followed by immersing in liquid N₂ to freeze.

1.4 Protocol for TCA Extraction

• 1. All extraction steps are carried out at 0–4°C (e.g., on ice).

• 2. Extract frozen or lyophilized powders (e.g., 30–40 mg) in microfuge tubes with 4–6:1 or 40–60:1 (v/w) volumes of 10% (w/v) ice-cold TCA, respectively. Vortex the mixture rigorously into a homogenous suspension. If homogenization is difficult by vortexing, it can be aided by a pellet pestle for microfuge tubes (cf. Fig. 7). If frozen powder is extracted, make sure that the homogenate is completely thawed before centrifugation.

  Fig. 7.

  

  Pellet pestle for microfuge tubes.

• 3. Centrifuge the homogenate in a refrigerated centrifuge (set at 4°C) at ≥15,000 rpm in a microfuge rotor for 20–30 min.

• 4. Recover the supernatant with a fine-tip transfer pipette and store on ice. It is important to avoid pipetting tissue powder into the supernatant.

• 5. Extract the residue again according to Step 2 and centrifuge according to Step 3.

• 6. Pool supernatants from the two extractions and lyophilize using a liquid N₂ pretrap to prevent TCA vapor from reaching the vacuum pump.

• 7. Store lyophilized extract at −80°C until further analysis.

1.5 Protocol for 60% Acetonitrile Extraction

• 1. Homogenize 5 mg of lyophilized tissue powder or cell mass in ≥0.4 mL (1:80 w/v) 60% acetonitrile (HPLC or higher solvent grade). Homogenization is achieved by rigorous vortexing or by mechanical shaking in glass beads using a micro ball mill (e.g., MM200, Retsch, Inc.).

• 2. Incubate the homogenate for 30 min at −80°C to facilitate precipitation of macromolecules.

• 3. Centrifuge the thawed sample at ≥15,000 g for 20–30 min at 4°C.

• 4. Recover supernatant using a fine-tip transfer pipette into a 2-mL polypropylene microfuge tube or a 7-mL scintillation vial.

• 5. Repeat steps 1–3 (the −80°C step can be omitted) and pool supernatants.

• 6. Lyophilize extract using a liquid N₂ pretrap to prevent acetonitrile vapor from reaching the vacuum pump.

• 7. Store lyophilized extract at −80 C until analysis.

1.6 Protocol for Chloroform/Methanol/Water Extraction

• 1. Homogenize 5 mg of lyophilized tissue powder or cell mass in 0.4 mL methanol in a 2-mL microfuge tube by rigorous vortexing or by mechanical shaking in glass beads for 1 min at 30 Hz using a micro ball mill (e.g., MM200, Retsch, Inc.). Alternatively, tissue or cell residue after polar metabolite extraction can be sequentially extracted with methanol for lipid components. For PCA- or TCA-extracted residues, remove acids with two to three times wash in nanopure water. All residues are lyophilized before lipid extraction.

• 2. Incubate the homogenate for 20 min at 60 C.

• 3. Cool and centrifuge sample at ≥15,000 g for 30 min at 4 C.

• 4. Recover supernatant using a fine-tip transfer pipette into a screw-cap glass vial.

• 5. Add 0.4 mL nanopure water and 0.2 mL chloroform and shake rigorously for 2 min.

• 6. Centrifuge the biphasic solvent mixture at ≥14,000 g for 10 min at 4°C to separate the two phases.

• 7. Recover the top aqueous layer using a fine-tip transfer pipette into a 2-mL microfuge tube or a 7-mL scintillation vial. Avoid transferring the protein pellet at the interface of the aqueous and chloroform layers.

Note: The bottom chloroform layer can also be recovered for nonpolar metabolites.

• 8. Lyophilize extract using a liquid N₂ pretrap to prevent solvent vapor from reaching the vacuum pump.

• 9. Store lyophilized extract at −80°C until analysis.

1.7 Protocol for Methanol Extraction of Lipids

• 1. Homogenize medium-free lyophilized cell mass or tissue powder (e.g., 1–5 mg) in 100% methanol (HPLC or MS grade) using a ratio of dry weight:methanol volume ≥100:1 plus 1 mM butylated hydroxytoluene (BHT, as antioxidant). Homogenization can be done by mechanically beating the pellet with glass beads in a micro ball mill (e.g., MM200, Retsch, Inc.) for 1 min at 30 Hz or by a pellet pestle in microfuge tubes.

• 2. Let the homogenate stand at room temperature in the dark for 30 min.

• 3. Centrifuge at ≥18,000 × g for 10–20 min at 4°C.

• 4. Repeat step 1 with another aliquot of 100% methanol and centrifuge as in step 2.

• 5. Pool both supernatants and dry under vacuum in a Speedvac or lyophilizer with a liquid N₂ pretrap.

• 6. Dissolve lipid residue in methanol or other appropriate solvents and store in Teflon-faced septum screw-cap glass vials at −80°C until analysis.

Note: Lipid extract should be filtered through 0.45-μM solvent-compatible filters (e.g., regenerated cellulose, Alltech Assoc.) prior to NMR or MS analysis.

Glossary

BHT

Butylated hydroxytoluene

CMW

Chloroform/methanol/water partition

FT-ICR-MS

Fourier transform-ion cyclotron resonance mass spectrometry

GC-MS

Gas chromatography-mass spectrometry

GSH

Reduced glutathione

GSSG

Oxidized glutathione

HSQC

Heteronuclear single quantum coherence spectroscopy

Isotopomer

Compounds of identical chemical structure but differing in isotopic composition at individual atoms

LC-MS

Liquid chromatography-mass spectrometry

MTBSTFA

N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide

PCA

Perchloric acid

TCA

Trichloroacetic acid

TOCSY

Total correlation spectroscopy