This protocol has many advantages over the currently used yeast metabolomics because it has high specificity, high sensitivity, and also it can profile many different classes of metabolites, including isomeric, isobaric, hydrophilic, and hydrophobic molecules. The quenching method used in this protocol stops all enzymatic reactions while reduces significantly the leakage of metabolites from the cells. The MS1 library built in this protocol is from the various MS2 runs.
After overnight culture at 30 degrees Celsius and 200 revolutions per minute, determine the number of the yeast cells per milliliter of culture and add five milliliters of sterilized 20%stock solution of glucose to each of two Erlenmeyer flasks containing 45 milliliters of autoclaved YP medium per flask. Use a sterile transfer pipette to add five times 10 to the seventh yeast cells to each flask and grow the yeast cells for at least 24 additional hours at 30 degrees Celsius at 200 revolutions per minute. For metabolite extraction, after culture, collect the yeast cells by centrifugation and quickly remove the supernatant.
Place the tube on dry ice and add two milliliters of minus 20 degrees Celsius chloroform, one milliliter of minus 20 degrees Celsius methanol, one milliliter of ice cold nano pure water, and 200 microliters of 425 to 600 micron acid washed glass beads to the cells. When the beads have been added, place the tube covered with aluminum foil into a foam tube holder kit with a retainer and vortex the sample for 30 minutes at medium speed at four degrees Celsius to facilitate metabolite extraction. Next, incubate the tube for 15 minutes on ice before centrifuging the sample to allow separation of the upper aqueous phase from the middle debris and protein and lower organic phases.
Use a micropipette to transfer approximately 400 microliters of the upper aqueous phase to a washed and labeled 1.5 milliliter tube containing 800 microliters of minus 20 degrees Celsius acetonitrile. Then centrifuge the sample and transfer 800 microliters of the upper portion of the supernatant to a labeled mass spectrometry vial for zero degree storage until liquid chromatography tandem mass spectrometry analysis. To separate the extracted metabolites, ultrasonicate the sample vial for 15 minutes, followed by three 10-second vortexes at room temperature.
After vortexing, place the vial into the well plate of the liquid chromatographer and set the column to 45 degrees Celsius with a flow rate of 0.25 milliliters per minute and the well plate to zero degrees Celsius. Then use the table to set the liquid chromatography gradients for the analysis. After separation, use 10 microliter sample volumes of the injection in both the electrospray ionization positive and negative modes in a mass spectrometer equipped with heated electrospray ionization to identify and quantify the water soluble metabolites.
At the end of the analysis, open the raw data in an appropriate compound analysis software program and utilize the mass spectral fragmentation library to match the mass spectra to annotate the metabolites identified in the analysis. The exact masses of the MS1 and isotope patterns can also be identified to allow annotation of the metabolites using online databases. Then use the library of databases and spectra to search for the MS2 spectra of the raw data.
The next day, after quenching the yeast cells, thoroughly wash the cells with 15 milliliters of ABC buffer and collect the cells by centrifugation. Resuspend the pellet in one milliliter of fresh ABC buffer and add 500 microliters of the propidium iodide solution to the cells. Vortex the sample three times for 10 seconds per vortex and incubate the cells for 10 minutes on ice protected from light.
At the end of the incubation, collect the cells by centrifugation and wash the cells three times with one milliliter of fresh ABC buffer per wash. After the last wash, resuspend the pellet in 300 microliters of fresh ABC buffer and add 10 microliters of the resulting cell suspension to a microscope slide. Use a fluorescence microscope to capture differential interference contrast and fluorescence microscopy images of the cells with the filters set to an excitation wavelength of 593 nanometers and an emission wavelength of 636 nanometers.
Then use an appropriate image analysis program to count the total cell number of cells in the brightfield and fluorescence images, and to determine the fluorescence intensity of staining for individual cells. The modified cell quenching method causes significantly lower damage to the plasma membrane and cell wall than the non-buffered 80%methanol at minus 40 degrees Celsius quenching method. Indeed, almost all of the cells subjected to quenching using the modified method exhibit red fluorescence emission, which is characteristic of yeast cells in which the plasma membrane and cell wall are not damaged.
In contrast, almost all of the cells subjected to quenching using the non-buffered 80%methanol at 40 degrees Celsius method displayed green fluorescence emission, which is characteristic of yeast cells in which the plasma membrane and cell wall are significantly damaged. The modified cell quenching method also causes significantly lower leakage of water soluble metabolites from yeast cells than the non-buffered 80%methanol at minus 40 degrees Celsius quenching method. The retention time shift values of water soluble metabolite standards are significantly lower and the peak shapes are substantially sharper for the zwitterionic phase column compared to the reverse phase column.
Another advantage of the liquid chromatography tandem mass spectrometry method is the ability to use the zwitterionic phase column to efficiently separate different water soluble metabolites with diverse structural, physical, and chemical properties. Make sure to do the propidium iodide standing in dark and also make sure to collect the upper phase while not disturbing the middle phase. If any MSP cannot be annotated due to the lack of the MS2 spectral library, then use an NMR to illustrate this structure.
