The overall goal of this procedure is to precisely analyze the amount and the structure of each Sphingomyelin species in biological samples. This method can help answer key questions in the lipid field pertaining to lipid biology and lipid The main advantage of this technique is that we can characterize a variety of Sphingomyelin species in biological samples by chromatography mass spectrometry system. To begin, remove the culture media from a 10 centimeter tissue culture dish containing HeLa cells and rinse the cells twice with six milliliters of ice cold PBS.
Then add one milliliter of the ice cold PBS into the 10 centimeter tissue culture dish and use a cell scraper to harvest the cells. Once removed from the dish's surface, transfer the cells into two milliliter siliconized plastic tubes. Following centrifugation, remove the supernatant and add one milliliter methanol to each cell pellet.
Then briefly vortex the tubes. Next, place the samples into a bath-type sonicator and sonicate at 200 watts for five minutes. When finished, transfer the whole cell homogenate into test tubes equipped with Teflon lined screw caps.
Now add one milliliter of methanol, one milliliter of chloroform, 0.8 milliliters of double distilled water in 50 microliters of 10 micromolar of an internal standard into each test tube. Cap the tubes and vortex them vigorously at 2, 500 rpm for five minutes at room temperature. Then add an additional one milliliter of chloroform and one milliliter of double distilled water to each tube.
Again, vortex the tubes vigorously at 2, 500 rpm for five minutes at room temperature. Next, centrifuge the samples in order to completely separate the aqueous and organic phases. Transfer the lower phase into disposable glass tubes.
Add two milliliters of chloroform into the test tubes. Then vortex the tubes vigorously at 2, 500 rpm for five minutes at room temperature to sufficiently mix with the upper phase and interphasial fluff. Following a second centrifugation, transfer the modified lower phase into the disposable glass tubes along with the initial lower phase.
Now place the glass tubes under a nitrogen stream for approximately 60 minutes and completely remove the organic solvents in the collected lower phase. Finally, reconstitute the samples with 500 microliters of methanol or ethanol. Filter the resulting mixture with a 0.02 micron filter into glass vials.
Then store the sample at minus 20 degrees Celsius. Perform serial dilutions of the standard compound and add 50 microliters of each concentration into separate test tubes. Next, prepare three quality control samples by adding an amount within three times the lower boundary of the standard curve to the first sample, an amount near the center of the curve for the second sample, and an amount near the upper boundary of the standard curve for the third sample.
Then add the cell homogenate in one milliliter of methanol into each test tube and extract the total lipid fraction from each sample using the method just shown. To begin, prepare the mobile phase. Mix together acetonitrile, methanol, and double distilled water at a two to two to one ratio for the aqueous phase.
Use isopropanol for the organic phase in glass bottles with Teflon lined screw caps. Then sonicate both of the mobile phases for five minutes in a bath-type sonicator. Then add formic acid to a final concentration of 26.4 millimolar and ammonium hydroxide at 14.9 millimolar into each mobile phase.
For qualitative analysis, activate the high performance liquid chromatography system. Put inlet tubes into the glass bottles that contain the mobile phases and purge the HPLC lines. Then link a C18 HPLC column to the HPLC system.
Keep the temperature in the column oven at 50 degrees Celsius and condition the column with the aqueous mobile phase at 100 microliters per minute. During the run, place the samples into a sample rack of the auto sampler. Set the parameters of the triple quadruple and quadruple linear ion trap mass spectrometry system for three-stage mass spectrometry analysis as listed in table one and table two of the accompanying text protocol.
Also, set the parameters for the first and second precursor ions of the SM species of interest as described in more detail in the accompanying text protocol. Create a batch file and submit the batch file to obtain the data of MS three product ion spectra of each Sphingomyelin species by liquid chromatography electrospray ionization tandem mass spectrometric analysis. Obtain the three-phase MS product ion spectra of the Sphingomyelin species of interest by liquid chromatography electrospray ionization tandem mass spectrometry analysis.
Then assign each MS three product ion spectra of the Sphingomyelin by comparing the mass to charge ratio of the product ion spectra in the exact mass of the Sphingoid long-chain base and n-acyl moiety of interest. Quantitatively analyze the Sphingomyelin using liquid chromatography electrospray ionization tandem mass spectrometry analysis as described in the accompanying text protocol. Then process the multiple reaction monitoring data using the software for data integration to obtain the data of the peak area for the extracted ion chromatogram of each Sphingomyelin species.
Quantify each Sphingomyelin species and construct the standard curves as described in the accompanying text protocol. The spectrum of both chemically synthesized Sphingomyelin and Sphingomyelin in lipid samples extracted from HeLa cells are shown here. Of note, the spectrum intensity of demethylated sphingosylphosphorylcholine is larger than that of the SM n-acyl moiety in both samples.
Also of interest, the spectrum corresponding to sphingosine-1-phosphate is useful to speculate the number of carbon and double bonds of the sphingoid long-chain base of the Sphingomyelin. In the present quantitative method, it is critical to precisely prepare samples for constructing the calibration curve in validating this method. The curve fitting up low concentration was clearly improved by using the weighting factor equals one over x squared as compared with that using the weighting factor of one or one over x.
This technique paved the way for researchers in the field of biology and industry to characterize a variety of Sphingomyelin species in biological samples and industry products such as cosmetics. After watching this video, you should have a good understanding of how to precisely analyze the amount and the structure of each Sphingomyelin species in biological samples. Don't forget that working with absorbents can be extremely hazardous and precautions such as using proper glass wears should always be taken while performing this procedure.
In addition, it is important to wear gloves to prevent the contamination of lipids derived from your hands.
