This protocol allows one to quickly analyze the major components of the yeast lipid do. It begins by harvesting and washing yeast cells. Washed cells are placed in a one to two chloroform, methanol mix, and lysed with glass beads.
Addition of chloroform and water separates the two phases. The organic phase is collected, dried, and the lipid film then dissolved in chloroform for storage prior to mass spectroscopy. The extracted lipids are diluted in chloroform and methanol with ammonium hydroxide and a mix of internal standards is added.
The lipid solution can now be injected directly into the mass spectrometer. Hi, I'm Simon Bork from the laboratory of Vladimir Teco at Concordia University's Department of Biology. Today I'm gonna show you a method for quantifying yeast lipids using mass spectroscopy.
We use this procedure in our laboratory to study changes of yeast lipid under different growth conditions. So let's get started. To begin this procedure, resus suspend the previously prepared frozen yeast pellets in 1.6 milliliters of ice, cold distilled water, and transfer 1.6 milliliters of the cell suspension to high speed glass centrifuge tubes.
Note that throughout this procedure, all manipulations with extracted lipids are done using glass pipettes or syringes. Since plastics will create a large background signal, if they come into contact with chloroform, start the lipid extraction by adding six milliliters of a chloroform methanol mix at a one to two ratio and 0.8 milliliters of glass beads measured in an einor tube to the cell suspension. The exact volumes are not critical as long as the ratio of one to two to 0.8 for chloroform, methanol, and yeast suspended in water is kept.Vortex.
The cell suspension and glass beads mix two times for one minute each time, continue by adding two milliliters of chloroform and mixing gently then incubate for five minutes at room temperature with occasional mixing. After the five minutes incubation, add two milliliters of distilled water and mix gently. Then again, incubate for five minutes at room temperature with occasional mixing.
Here also, it is more important to maintain the two to two to 1.8 ratio for chloroform, methanol, and water rather than a specific volume. When the second incubation is complete. Centrifuge for five minutes at 3000 Gs at room temperature, collect all of the supernatant from this first extraction into a new high speed glass centrifuge tube and add 3.2 milliliters of chloroform to the remaining cell pellets For a second lipid extraction.
Vortex the cell pellets with the added chloroform two times for one minute each time. Then centrifuge for five minutes at 3000 Gs At room temperature, add the supernatant from this second extraction to the supernatant collected after the first extraction. Next centrifuge the collected supernatants for five minutes.
At 3000 Gs at room temperature, the extracted lipids are now dissolved in the lower organic phase. Discard the upper aqueous phase and transfer the lower phase into a new high speed glass centrifuge tube. To produce cleaner spectra on the mass spectrometer centrifuge, the organic phase for five minutes at 3000 Gs at room temperature and to transfer the entire supernatant into a glass vial.
Dry the dissolved lipids under nitrogen, dissolve the dried lipid film in 500 microliters of chloroform and store at minus 20 degrees Celsius until ready to analyze the samples by mass spectrometry. Start by preparing a standard mix of the lipids in chloroform as listed in table one found in the accompanying written protocol. Each lipid represents a different class.
This solution can be stored at minus 20 degrees Celsius for use later prior to injection of the extracted lipids into the mass spectrometer. Combined 10 microliters of the sample with 10 microliters of the standard mix of lipids. Note that the standard to sample ratio can be changed as needed.
Resolve the sample and standard lipids mix using a micro mass Q to F two mass spectrometer, equipped with a nano electrospray source operating at a flow rate of one microliter per minute. The settings are detailed in table two of the accompanying written protocol. Although the use of the high resolution of a Q to F mass spectrometer is advantageous, it is not a mandatory requirement.
Exact instrument parameters will vary between mass spectrometers. After data acquisition, the mass spectra are smoothed, the background subtracted and the peaks are centered. Finally, export the peak list to excel.
Then normalize the peaks of each lipid glass to their internal standard. Depending on the application, it may be necessary to perform further post-processing such as de isotope and deconvolution. This method allows the rapid in-depth determination of the major components of the yeast lipid do.
Here we have highlighted one lipid, which has been de isotope de convoluted and normalized to its internal standard. We've just shown you how to isolate yeast lipids and how to determine the yeast lipid on using electros display ionization mass spectroscopy. When doing this procedure, it's important to remember to avoid the use of plastics and to keep your sample in the linear range of concentrations.
This method is quite flexible and in many of the volumes can be changed as needed. This extraction protocol is also suitable for other methods of lipid analysis, such as thin layer chromatography and multidimensional mass spectroscopy. So that's it.
Thanks for watching and good luck with your experiments.
