Stable Isotope Metabolomics Extraction Protocol

This protocol details metabolite extraction from coral larval samples labeled with stable isotopes (C13 bicarbonate) and control samples (C12 bicarbonate).

Protocol modified from Kevin Wong’s metabolite extraction protocol here.

Equipment Needed

  • 2 mL glass dounces
  • 11mm snap cap auto sampler vials - Thermo Scientific 250 µL, No. C4011-13
  • 11mm snap cap auto sampler caps = Thermo Scientific Part No. 501-313, Orange snap PTFE/SIL
  • LC-MS grade Methanol
  • LC-MS grade Acetonitrile
  • LC-MS grade Formic Acid
  • LC-MS grade water
  • Tissue Tearor or other probe homogenizer
  • 10% bleach
  • 70% ethanol
  • DI water

CRITICAL: Autoclave all glassware and equipment prior to protocol, preparing reagents, and between batches of samples.

Prepare Reagents

Extraction buffer

This extraction buffer is a ratio of 40:40:20 of methanol, acetonitrile, and water, with 0.5%[v/v] Formic Acid

For a total of 200mL extraction buffer:

80mL methanol
80mL acetonitrile
40mL water
1mL formic acid

Store at 4°C in a non-flammable fridge

15% ammonium bicarbonate solution

The solubility of Ammonium bicarbonate in water is: 220g/L for 100%

To make a 250mL solution of 100% ammonium bicarbonate add 55g dry Ammonium bicarbonate in 250mL LCMS water.

Then, to make the 15% ammonium bicarbonate solution (250mL), add 37.5mL of the 100% Ammonium Bicarbonate solution in 212.5mL LCMS water.

Store at 4°C in a non-flammable fridge.

Separating Host, Holobiont, and Symbiont fractions

Prior to metabolite extraction, separate host, symbiont, and holobiont fractions for further extraction as described in our protocol here.

Before starting, label the desired tubes. For each biological sample (indicated by #), you will need to label one of each of the following tubes:

1.5mL tubes: “#-Host” “#-Sym” “#-Holo”

  1. Thaw sample (2mL tube) on dry ice
  2. Add 200uL of ice cold DI water
  3. Homogenize with motor for 45 sec-1 min on ice
  4. Cap the sample and place on ice.
  5. -CRITICAL- Clean the motor with 10% bleach, 70% ethanol, and DI water after each sample.
  6. Remove 100uL of the homogenized holobiont and place into new tube (#-Holo).
  7. Store on dry ice for further processing or put into -80C depending on samples you need to extract.
  8. Remove the remaining homogenate and place into a new tube (#-Sym).
  9. Spin the “Sym” tube at 2000g for 90 seconds at 4C.
  10. Remove the supernatant and place into a new tube (#-Host).
  11. Store on dry ice for further
  12. processing or put into -80C depending on samples you need to extract.
  13. The “Sym” tube now only contains a symbiont pellet.
  14. Add 100uL of DI water to the tube with the symbiont pellet and pipette up and down to mix
  15. If necessary, move the symbiont homogenate into a new tube.
  16. Store at -80C if not used for extractions.
  17. Samples can be stored in the “Pre-Processing” freezer box.

See sections below for extraction with protocol variations for each fraction.

Metabolite extraction

Prior to starting, label the following tubes for each sample fraction to intend to extract. Here, # indicates the biological sample ID and “Fraction” indicates the fraction (host, symbiont, or holobiont). Refer to sample list for desired fractions for extraction.

1.5mL tubes:
“#-Fraction-A”
“#-Fraction-B”

Autosampler vials: “#-Fraction-A Date Initials”
“#-Fraction-B Date Initials”

  1. Thaw chosen sample on dry ice if using frozen homogenized fractions. If proceeding directly after homogenization, immediately proceed with sample of interest in the steps below.
  2. Add sample to the the ice cold douce with 500uL of ice cold extraction buffer. Sit on dry ice for 5 minutes.
  3. If extracting the HOST or HOLOBIONT fractions, pipette the liquid fraction obtained from separations into the dounce.
  4. If extracting from the SYM fraction, add 500uL of the ice cold extraction buffer directly to the Sym 1.5mL tube. Use a pipette to gently resuspend the symbiont pellet. Then transfer the extraction buffer and suspended symbiont pellet to the dounce.
  5. Homogenize the sample on dry ice for ~1 minute with the douce.
  6. Transfer extract to a new 1.5 mL tube (Tube #-Fraction-A).
  7. Centrifuge for 10 minutes at 15000g at 4C
  8. Remove 500uL of the supernatant to a new 1.5 mL tube (Tube #-Fraction-B)
  9. Add 44uL of 15% ammonium bicarbonate
  10. Vortex and spin down
  11. Add 100uL from Tube B into duplicate labeled autosampler vials (#-Fraction-A and #-Fraction-B)
  12. Store the two 1.5 mL extract tubes (Tubes A and B) and the two autosampler vials at -80C in the “Post-Extraction” freezer box.

Samples are now ready to be shipped for metabolite quantification at Rutgers Shared Metabolomics Resources.

Written on April 8, 2022