This post details metaboloimcs extraction protocols for the 2023 Moorea Symbiotic Exchange project, which contains both larval and recruit samples.

Metabolomics Extraction Protocol

This post details metabolomics extraction protocols for samples taken during the 2023 Symbiotic Exchange project.

Samples

Samples include snap frozen recruits and larvae. We are processing 57 total samples for this project. The samples include C13 incubated samples as well as C13 dark-incubated samples and C12 control samples.

Materials

Chemicals:

• LCMS/HPLC grade water
• HPLC grade Methanol
• Acetonitrile
• Formic acid
• Ammonium bicarbonate (solid)

Supplies:

• Microtip sonicator for homogenization
• Centrifuge and rotor capable of spinning at 15,000 rcf and chilling to 4°C
• Sample tube freezer boxes (4 boxes)
• Plastics
  • 4 1.5 mL microcentrifuge tubes per sample, labeled
  • 2 Autosampler vials per sample, labeled
• 70% ethanol, 100% ethanol
• 10% bleach
• DI water
• P1000, P200 pipettes
• Standard consumables (gloves, KimWipes, etc.)
• Glass dounces

Protocol

Proceed with samples in the following order:

1. C12 samples
2. C13 dark samples
3. C13 samples in random order

Prepare tubes

Prepare labeled tubes for samples 1-57.

Homogenization/preparation

1.5 mL tubes = 2

”#” (homogenization) “#-Host” (host fraction)

Extraction

1.5 mL tubes = 2

”#-1” “#-2”

Autosampler vials = 2

”#-A Huffmyer” “#-B Huffmyer”

Sample prep, homogenization, and fractionation: Recruits

• Get small batches of samples from the freezer and hold on dry ice in a cooler

• Using cleaned (using ethanol follwed by bleach) clippers, remove a >1 cm2 area of tissue and place into a labeled 1.5 mL tube.

• Add 1000 µL ice cold LCMS grade water. Return to the freezer at this step if needed if not proceeding with homogenization.

• Place on dry ice between all steps.

• Sonicate sample for 10 sec at 75% amplitude .

• Wipe the sonicator tip with 70% ethanol, and DI water after each sample.

• Spin at 3,000g for 5 min at 4°C.

• Remove supernatant and place in a new labeled tube (“host” tube). Record volume. If needed repeat a second time.

• Place in a freezer box and store at -80°C until extraction.

Sample prep, homogenization, and fractionation: Larvae

• Get small batches of samples from the freezer and hold on dry ice in a cooler

• Add 200 µL ice cold LCMS grade water to the original sample tube. Pipette into a new labeled tube. Return to the freezer at this step if needed if not proceeding with homogenization.

• Place on dry ice between all steps.

• Sonicate sample for 10 sec at 75% amplitude .

• Wipe the sonicator tip with 70% ethanol, and DI water after each sample.

• Spin at 3,000g for 5 min at 4°C.

• Remove supernatant and place in a new labeled tube (“host” tube). Record volume. If needed repeat a second time.

• Place in a freezer box and store at -80°C until extraction.

Prepare extraction buffers and supplies

Equipment

Before each batch, thoroughly clean glassware (dounces).

• After use, rinse with DI water thoroughly followed by a thorough rinse with ethanol.

• Rinse thoroughly with DI water.

• Repeat an ethanol rinse and DI water rinse.

• Do a final rinse with LCMS grade water.

• Dry on a clean surface.

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 200 mL extraction buffer:

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

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

15% Ammonium Bicarbonate

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

• 100% Ammonium Bicarbonate solution (250 mL)
  • 55g dry Ammonium bicarbonate in 250 mL LCMS water
• 15% Ammonium Bicarbonate Solution (250 mL)
  • 37.5 mL of 100% Ammonium Bicarbonate Solution + 212.5 mL LCMS water

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

Metabolite extraction

• Work in batches of 10 with the number of dounces available.

• Keep samples on dry ice and place dounces over dry ice on a tube rack in a cooler.

• Add sample (pipette from tube) into the ice cold douce with 500 µL of ice cold extraction buffer. Sit on dry ice for 5 minutes.

• Homogenize for 1 minute on dry ice in the dounce using the glass dounce.

• Transfer the liquid into a new 1.5 mL tube (“#-1”).

• Centrifuge for 10 minutes at 15,000g at 4°C.

• Remove 500 µL of the supernatant into a new 1.5 mL tube (“#-2”).

• Add 44 µL of 15% ammonium bicarbonate.

• Vortex and spin down in a mini centrifuge.

• Add 100 µL from tube “#-2” into duplicate labeled autosampler vials (“#-A Huffmyer” and “#-B Huffmyer”.

• Store the tubes (“Extraction extras box”) and autosampler vials (“Metabolite extracts box”) in the freezer at -80°C.

Sample storage and shipment

• Parafilm and secure the “Metabolite extracts box” with autosampler vials. Clearly label with name, date, and experiment.

• Coordinate with facility to ship samples using overnight FedEx shipment.

• Send in a box with dry ice (~8 lbs).

• Once data is obtained, discard of other extraction tubes.