This method can answer key questions in the metabolism field, such as how the oncometabolite L2-hydroxyglutarate is synthesized in healthy cells. The main advantage of this technique is that it allows the user to directly measure over 100 metabolites in a sensitive and reproducible manner. Generally, individuals new to this method will struggle because the protocol requires efficient sample processing and is extremely sensitive to water vapor.
To begin the protocol, add one milliliter of ice-cold 0.9%sodium chloride into a 1.5-milliliter tube containing previously prepared larvae. Close the lid, vertically flip the tube to thoroughly wash the larvae, and place the tube on ice for 30 seconds. The larvae will sink to the bottom of the tube, but the yeast will remain in suspension.
Once all larvae have formed a loose pellet, remove the sodium chloride solution using a one-milliliter pipette. Centrifuge the samples at 2, 000 g for one minute at four degrees Celsius. Remove all residual solution using a 200-microliter pipette, and immediately freeze the sample in liquid nitrogen.
Use an ethanol-proof marker to label the side of a two-milliliter screw-cap bead tube. Next, tare the mass of the labeled bead tube using an analytical balance capable of accurately measuring 0.01 milligrams. Use long forceps to remove the 1.5-milliliter sample tube from the liquid nitrogen dewar.
Wearing nitrile gloves, grab the frozen tube, invert it, and sharply pound the tube lid against the benchtop to dislodge the frozen pellet. Immediately pour the pellet into a pre-tared two-milliliter screw-cap bead tube. The sample must be quickly processed to ensure that endogenous metabolic pathways are not reactivated.
Quickly measure the combined mass of the larval pellet and bead tube. Immediately place the sample tube into liquid nitrogen. Place the sample tubes in a negative 20 degree Celsius benchtop cooler.
Add 0.8 milliliters of pre-chilled 90%methanol containing two micrograms per milliliter of succinic-D4 acid into each tube. Return the samples to the negative 20 degree Celsius benchtop cooler. Set up a negative control by adding 0.8 milliliters of pre-chilled 90%methanol containing two micrograms per milliliter of succinic-D4 acid into an empty bead tube.
Then, homogenize the samples for 30 seconds at 6.45 meters per second using a bead mill homogenizer located in a four degree Celsius temperature control room. Return the homogenized samples to the negative 20 degree Celsius benchtop cooler, and transfer the cooler into a negative 20 degree Celsius freezer for at least one hour. After incubating, centrifuge the tubes at 20, 000 g for five minutes at four degrees Celsius to remove the resulting precipitate.
Transfer 600 microliters of the supernatant into a new 1.5-milliliter microcentrifuge tube, and do not disturb the precipitate. Open all the sample tubes, and place them in a vacuum centrifuge. Dry the samples at room temperature until all solvent is removed.
If the dried samples were stored at negative 80 degrees Celsius, place unopened sample tubes in a vacuum centrifuge and dry them for 30 minutes. Prepare a solution of 40 milligrams per milliliter methoxylamine hydrochloride, or MOX, in anhydrous pyridine. Store the MOX and pyridine in a desiccator.
Next, use a heat gun to dry a one-milliliter glass syringe for about five seconds. Insert the needle of the syringe into the bottle of anhydrous pyridine. Remove one milliliter of pyridine, and add it to a microfuge tube containing 40 milligrams of MOX.
This portion of the protocol is extremely sensitive to water. All reagents must be stored in a desiccator prior to use. In addition, the user must ensure that the sample has minimal exposure to atmospheric water vapor.
Then, flush the bottle of anhydrous pyridine with argon. Seal the bottle, and return it to the desiccator. Dissolve the MOX in pyridine by incubating the tube in a thermal mixer at 35 degrees Celsius for 10 minutes at 600 rpm.
Next, add 40 microliters of 40 milligrams per milliliter MOX in anhydrous pyridine solution to the dried sample. Vortex the sample for 10 seconds, and briefly centrifuge it. Then, incubate the sample in a thermal mixer.
Centrifuge the sample at 20, 000 g or maximum speed for five minutes to remove the particle matter. Transfer 25 microliters of the supernatant into an autosampler vial with a 250-microliter deactivated glass microvolume insert. Add 40 microliters of MSTFA containing 1%TMCS.
Place a cap on the autosampler vial, and seal the vial using a crimper tool. Incubate the sample at 37 degrees Celsius for one hour with shaking at 250 rpm. Finally, add three microliters of previously prepared fatty acid methyl ester standard, or FAMES, solution, to the autosampler vial using a robotic autosampler immediately before injection.
Gas chromatography-mass spectrometry of lactate dehydrogenase mutant larvae exhibit significant changes in lactate, pyruvate, and L2-hydroxyglutarate compared with control larvae. The GC-MS spectra generated with the protocol show many visible features and a notable peak for trehalose, which normally represents the largest peak in a larval sample. Principle component analysis, or PCA, clearly shows that the knockout and wild-type groups separate from each other and that there is no outlier in either group.
While attempting this procedure, it's important to remember to keep the samples frozen prior to homogenization and to keep all reagents dry throughout the procedure.
