This protocol provides an accurate and reproducible method for quantifying ten different lipid classes of Saccharomyces cerevisiae using a single extraction method and a single LC-MS method. This method allows the detection and quantification of isobaric and isomeric lipidic species having different chemical and physical properties. For Saccharomyces cerevisiae culture, first add 5 ml of sterile 20%glucose stock solution to each of two Erlenmeyer flasks containing sterile YNB medium to a final concentration of 2%glucose.
Then, use a sterile pipette to transfer a volume of an overnight yeast culture containing five times ten to the seventh yeast cells into each flask and culture the flasks for at least 24 hours at 30 degrees Celsius with rotational shaking at 200 revolutions per minute. For lipid extraction, determine the total number of yeast cells per milliliter of culture and transfer five times ten to the seventh cells into a 15 ml centrifuge tube. Add ice cold nano-pure water to bring the total volume up to 15 ml and collect the cells by centrifugation.
Re-suspend the yeast pellet in 15 ml of ice cold iso-tonic ammonium bicarbonate buffer for a second centrifugation and re-suspend the pellet in 1.5 ml of fresh ice cold ammonium bicarbonate solution. At the end of the centrifugation, store the pellet at 80 degrees celsius until the extraction. To begin the lipid extraction, thaw the cell pellet on ice before re-suspending the cells in 200 microliters of ice cold nano-pure water.
In a fume hood, transfer the cell suspension to a 15 ml, high strength glass, screw top centrifuge tube with a polytetrafluoroethylene lined cap and add 25 microliters of an internal lipids standards mixture, prepared in 2:1 chloroform methanol. 100 microliters of 425 to 600 micromolar acid washed glass beads and 600 microliters of a 17:1 chloroform methanol to the tube. Vortex the tube at high speed for five minutes at room temperature to disrupt the cells.
Followed by vortexing for one hour at low speed at room temperature to facilitate lipid extraction. At the end of the extraction, incubate the sample for 15 minutes on ice, to promote protein precipitation and the separation of the aqueous and organic phases. At the end of the incubation, centrifuge the sample to further separate the upper aqueous phase from the lower organic phase and, in the fume hood, use a borosilicate glass pipette to transfer the lower organic phase to a new 15 ml high strength glass screw top centrifuge tube with a polytetrafluoroethylene lined cap.
Place the lower organic phase under the flow of nitrogen gas and, in the fume hood, add 300 microliters of a 2:1 chloroform methanol mixture to the remaining upper aqueous phase. To facilitate phosphatidicacid, phosphatidylserine, phosphatidylinositol and cardio-lipid extraction vortex the tube vigorously for five minutes at room temperature before centrifuging the sample. Use a borosilicate glass pipette to transfer the lower organic phase into the lower organic phase under nitrogen and evaporate the solvent in the combined organic phases in the fume hood.
Then, close the tubes of lipid film under the flow of nitrogen gas and store the samples at 80 degrees celsius. For liquid chromatography, separation of the extracted lipids at 500 microliters of a 63:35:5 aceto-nitrile-2-propanol nano-pure water mixture to the tube of lipid film and vortex three times for ten seconds per vortex at room temperature. Subject the tube contents to ultrasonic sonication for 15 minutes before vortexing the tube three times for ten seconds per vortex as just demonstrated.
Transfer 100 microliters of the sample into a glass vial with an insert used for a 24 well plate and eliminate any bubbles within the insert. Place the insert into one well of a 24 well plate and load a reverse phase C18 column CSH coupled to a pre-column into the liquid chromatography system. Set the column temperature to 55 degrees celsius and the flow to 300 microliters per minute.
When all of the parameters have been set, load ten microliters of an extraction blank before loading the first sample. Then, separate the different lipid species using the liquid chromatography gradient, as indicated. A representative total ion chromatogram from the LC-MS data for the lipids extracted from the cells will be generated.
For mass spectrometric analysis of the extracted lipids, load the samples onto a mass spectrometer equipped with a heated electro-spray ionization ion source and set the analysis parameters as outlined in the table. Use the Fourier Transform Analyzer to detect MS-1 parent ions at a resolution of 60, 000 and within the mass range of 150-2000 daltons. Then, detect the MS-2 secondary ions using the settings as indicated in the table.
To identify and quantify different lipids from broad tandem LC-MS files, search LC-MS raw files containing full scan MS-1 data and data dependent MS-2 data for free, unesterified fatty acids, cardio-lipid, phytocerimide, phytosphyngocene, phosphatidylcholine, phosphatidylethanolamine, phosphatydlglycerol, phosphatydlinositol, phosphatydlserine and triacylglycerol lipid classes using a mass divided by charge tolerance of five parts per million for precursor ions and ten parts per million for product ions. Then, follow the instructions provided in the software user manual to identify internal lipid standards and lipid species with unusual fatty acid compositions. This sensitive tandem LC-MS method enables the identification and quantification of molecular species of lipids at concentrations as low as 0.165 picomolar per microliter.
This limit of quantification differs from different lipid classes within a wide range of concentrations. This method can be used to increase the efficiency of ionization for lipids by using alternative mobile phase additives for the electrospray ionization mass spectrometry. Each of these alternative mobile phase additives can be used for both the normal phase and reverse phase LC columns.
The collision induced dissociation method is beneficial if used in combination with the ammonium acetate mobile phase additive for the electro-spray ionization negative mode of mass spectrometry, as, under these conditions, it allows the increase in the efficiency of MS-1 lipid ion fragmentation into MS-2 products. In contrast, the high energy, collisional dissociation method is favorable if used in combination with the ammonium formate mobile phase additive for the electro-spray ionization positive mode of mass spectrometry as under these conditions, it enables an increase in the efficiency of MS-1 lipid ion fragmentation into MS-2 products. Do not mix the phases while collecting the organic phase.
Close the tubes under the flow of nitrogen gas. Eliminate any bubble within the insert and connect the column in the correct direction. If the identity of any peak cannot be determined by LC-MS, MS-NMR can be used to deduce the structure of that molecule.
