The overall goal of this procedure is to quantitatively investigate the metabolism of multiple nutrient sources using labeled substrates. This workflow allows the fate of consumed nutrients and the metabolic origin of compounds synthesized by yeasts to be determined. This method can help to answer key questions in the microbial metabolism field such how all the metabolism operate depending on environmental conditions.
The main advantage of this technique is that it allow the elucidation and quantification of the protein of nutrient distribution from multiple sources. This method was used to investigate the nitrogen metabolism of yeast, but it can also be applied to other metabolisms and microorganisms. The key to success with this experiment lies in the reproducibility of fermentations.
Users must pay particular attention to collect the cells in the same defined physiological stage. Demonstrating the procedure will be Christian Picou from our technical staff, Pauline Seguinot, a PhD student from the laboratory, and Stephanie Rollero, a post-doctorate fellow. In an autoclave, pasteurize each synthetic medium prepared for each nitrogen source in a one-liter flask containing a magnetic stir bar.
Dissolve the appropriate amount of labeled molecule to reach the desired final concentration in the medium. Sterilize the medium using a disposable vacuum filtration system. Using a sterile measuring cylinder, divide the medium between two pre-sterilized fermenters that contain a magnetic stir bar and are equipped with fermentation locks to avoid the entry of oxygen but allow the release of carbon dioxide.
After growing a cerevisiae strain in YPD medium, collect a pre-culture aliquot under laminar flow and quantify the cell population using an electronic particle counter that is fitted with a 100-micrometer aperture. After centrifuging the pre-culture, suspend the pellet in an appropriate volume of sterile water to obtain a final concentration of 2.5 times 10 to the 9th cells per milliliter. Then, inoculate each fermenter with one milliliter of the cell suspension.
Following this, prepare a fermentation platform by installing the fermenters in the support guides that are properly placed on the 21-position stirring plates and set the stirring rate at 270 RPM. To start the online monitoring of each fermentation, launch the robot control application, click the Start Assay button and select the fermentation volume to be carried out. To ensure that the displayed interface permits the indication of the number and the position of fermenters on the platform, right-click on the slot location and choose Disable to inactivate the monitoring of the empty positions.
To initialize the calculation software, click on the Initializer button and validate with OK.Then, click on the Start button of the robot control application to start the weight acquisitions. Next, centrifuge two six-milliliter samples taken from the fermenters at 2, 000 times G for five minutes at four degrees Celsius. When finished, transfer each frozen supernatant into two tubes and store at minus 80 degrees Celsius.
Wash each pellet twice with five milliliters of distilled water and store at minus 80 degrees Celsius for the measurement of isotopic enrichment. Add 200 microliters of a 25%sulfosalicylic acid solution that contains 2.5 millimolar norleucine as an internal standard to 800 microliters of sample to remove molecules with high molecular weights. After incubation and centrifugation, filter the mixture through a 0.22-micrometer pore-size nitrocellulose membrane and place the sample in an amino acid analysis system.
In the program or software of the analysis system, click on the button Run to begin the liquid chromatography analyses with the analyzer equipped with a cation exchange column. Next, prepare an oxidized extract by suspending a previously prepared labeled cell pellet in 200 microliters of performic acid. After incubating for four hours at four degrees Celsius, stop the reaction by adding 33.6 milligrams of sodium sulfate.
Add 800 microliters of six normal hydrochloric acid to the oxidized extract, and incubate the sample in a hermatically-sealed glass tube for 16 hours at 110 degrees Celsius in a dry-heat oven. After allowing the sample to cool to room temperature, add 200 microliters of 2.5 millimolar neuroleucine and remove the hydrogen chloride gas with a stream of nitrogen. Wash the dried extract twice with 800 microliters of distilled water, and then 800 microliters of ethanol.
Then, add 800 microliters of 200-millimolar lithium acetate buffer to the extract. Determine the relative concentrations of amino acids within proteins in the sample using the chromatographic method previously described. Hydrolyze a labeled cell pellet by adding 1.2 milliliters of six-molar hydrochloric acid and incubating the sample for 16 hours at 105 degrees Celsius in a tightly-closed glass tube in a dry-heat oven.
After allowing the sample to cool to room temperature, add 1.2 milliliters of distilled water. Following centrifugation to remove the cellular debris, distribute the supernatant into six 400-microliter fractions in open glass tubes. Dry the fractions in the dry-heat oven at 105 degrees Celsius for four to five hours until they reach the consistency of syrup.
During the biomass hydrolysis, dry the fraction until the consistency of a syrup by frequently controlling the acid of operation. For ECF derivatization, dissolve the cooled dried hydrolysate in 200 microliters of 20-millimolar hydrochloric acid and 133 microliters of a one-to-four mixture of pyridine and ethanol. Add 50 microliters of ethyl chloroformate to derivatize the amino acids.
After the carbon dioxide has been released, transfer the mixture to centrifuge tubes that contain 500 microliters of dichloromethane to extract the derivatized compounds. Vortex the samples for 10 seconds. After centrifuging for four minutes at 10, 000 times G, collect the lower organic phase of each sample with a Pasteur pipette and transfer to a GC autosampler vial that contains a conical glass insert so that each sample may be directly injected into the GCMS instrument.
HPLC derivatization reagents are used for the ECF procedure. Change frequently the ECF reagents. For DMF-DMA derivatization, dissolve the dried hydrolysate in 50 microliters of methanol and 200 microliters of acetonitrile.
Add 300 microliters of DMF-DMA and vortex the sample. Then, transfer the sample to a GC autosampler vial that contains a conical glass insert. For BSTFA derivatization, suspend the hydrolysate in 200 microliters of acetonitrile.
Add 200 microliters of BSTFA to the sample and hermatically close the glass tube. After incubation for four hours at 135 degrees Celsius, transfer the cooled extract directly to a GC autosampler vial that contains a conical glass insert. Now, analyze the samples with a gas chromatograph equipped with an autosampler injector and coupled to a mass spectrometer.
Click on Instrument in the menu of the computer software. Under the Sequence tab, click the Edit Sample Log Table to create a sample list, and click Run and Run Sequence to start the injections. After the analyses are complete, record a cluster of intensities for each amino acid that corresponds to its different mass isotopomers.
Add 10 microliters of the appropriate deuterated internal standards to five milliliters of supernatant in a 15-milliliter glass tube. Add one milliliter of dichloromethane, tightly close the tube and shake the sample on a rocking platform for 20 minutes. After centrifugation, collect the lower organic phase in a 15-milliliter glass tube.
After repeating the dichloromethane extraction, dry the organic extract over 500 milligrams of anhydrous sodium sulfate and collect the liquid phase with a Pasteur pipette. Concentrate the extract by a factor of four under nitrogen flux. Then, transfer the concentrated sample to a GC autosampler vial.
For GCMS quantification, inject two microliters of sample with a split ratio of 10-to-one, and separate the extracted volatile molecules using the appropriate oven temperature profile. Using the spreadsheets in the text protocol, enter the raw data values that correspond to the concentration of extracellular amino acids, cell dry weight, protein content of cells, concentration of volatile molecules and isotopic enrichment of proteinogenic amino acids and volatile molecules. A schematic diagram of the workflow to investigate the management by yeast of the multiple nitrogen sources that are found during wine fermentation is shown here.
For different points of sampling, the biological parameters show a high reproducibility among fermenations. An overview of the redistribution of nitrogen from the sources in grape juice to all the proteinogenic amino acids is shown here. Combined with the determination of the mass balances, the measurement of the isotopic enrichment of proteinogenic amino acids provided the first quantification of the contribution of nitrogen-originated arginine, glutamine, ammonium and other sources to the amine groups of each of these compounds.
A comparison between the amount of consumed amino acid that is directly recovered into the biomass is depicted here. Partitioning of the consumed aliphatic amino acids, leucine and valine in the metabolic network in yeast is shown here. More than 96%of the consumed valine and leucine are recovered in their conversion products.
Surprisingly, a substantial fraction of valine and leucine are catabolized despite a considerable imbalance between the availability of these compounds in the medium and their content in biomass. Once mastered, this technique can be done in five days. While attempting this procedure, it's important to remember to carefully select the labeling part of the substrate according to the scientific issues.
To ensure a good biological reproducibility, perform the fermentation and chromatographic analysis in duplicate. After its development, this technique paved the way for researchers in microbiology field to explore how the metabolism and physiology of microorganism change depending on the environmental conditions. Don't forget that working with derivatization reagents and arginine solvent can be extremely hazardous, thus precautions such as wearing gloves and working under a fume hood should always be taken while performing this procedure.
