The overall goal of isolating lipid droplets for quantitative mass spectrometry is to understand the function of these organelles, for example, in the replication of pathogens such as the hepatitis C virus. This method can answer key questions in the infectious disease field such as why pathogens tie to the droplets for replication. The isolation of lipid droplets can be done in six to seven hours.
And during this procedure, it's important to avoid the contamination of keratin. To begin, remove 50 milliliters from each bottle of DMEM medium and add 50 milliliters of dialyzed fetal calf serum. Next, dissolve 50 milligrams of carbon 13 labeled lysine and 50 milligrams of carbon 13 labeled arginine in one milliliter of medium.
Mix thoroughly, and add the amino acids to the DMEM plus FCS medium. Then add PenStrep and glutamine substitute. Sterile filter the medium using a 0.45 micron filter, and label the bottle as heavy SILAC medium.
To prepare the light medium, repeat these steps using 50 milligrams of arginine and 50 milligrams of lysine. Label the bottle as light SILAC medium. Trypsinize the cells, and split into two wells of a six well culture plate.
One well should contain two milliliters of the heavy SILAC medium, and the other, two milliliters of the light SILAC medium. Culture the cells for at least six passages. After six passages, the incorporation of the heavy amino acids should be more than 95%Harvest one million cells of the heavy and light labeled cell populations to analyze the incorporation efficacy.
After the washing the cells in 1X PBS, pellet them by centrifugation for five minutes at 160 times g and four degrees Celsius. Re-suspend the cell pellets in 150 microliters of MS-buffer. Next, incubate the cells on ice for 30 minutes.
Then, lyse the cells by sonication at four degrees Celsius. Remove the cell debris by centrifugation for 10 minutes at 11, 000 times g and four degrees Celsius. Transfer the supernatant to a new tube, and determine the protein concentration with a detergent compatible protein assay.
Then, mix 75 micrograms of protein with six x sample buffer. After boiling at 95 degrees Celsius for five minutes, separate the proteins by SDS-PAGE in SDS running buffer at 180 bolts for approximately one hour. After Coomassie staining, excise the same protein band and analyze the incorporation efficacy by MS analysis.
Infect the light population of cells with the hepatitis C reporter virus by incubating them with fibrous stocks for four hours at 37 degrees Celsius. Expand HCV infected light cells and non-infected heavy cells as described in the text protocol. If using an HCV strain that carries a blasticidin resistance gene, add 10 micrograms per milliliter of blasticidin S to the light medium to select for HCV positive cells.
One day prior to lipid droplet isolation, wash the cells with PBS, tryspinize, and re-suspend in the corresponding light or heavy medium. Count the cells using a Neubauer counting chamber. Then, seed seven million cells of each population in a 150 square centimeter cell culture dish.
Prepare at least five dishes per light and heavy cell population. Culture the cells in 30 milliliters of medium per dish overnight. The next day, remove the medium, and wash the cells in 1X PBS.
Detach the cells in 1X PBS using a cell scraper. After counting both cell populations as before, pull equal cell numbers in 50 milliliter centrifuge tubes. Pellet the cells by centrifugation for five minutes at 160 times g and four degrees Celsius.
Remove the PBS, and re-suspend the cell pellet in one milliliter of sucrose buffer. Transfer the cell suspension to a tight-fitting Ounce homogenizer before the lysing the cells with 200 strokes on ice. Then, transfer the lysate into a 1.5 milliliter microfuge tube, and spin down the nuclei and cell debris for 10 minutes at 1, 000 times g and four degrees Celsius.
After centrifugation, store an aliquot of 25 microliters of the post-nuclear fraction, or PNS, at minus 20 degrees Celsius as an input control. Place the rest of the PNS fraction at the bottom of the centrifuge tube, and overlay with approximately three milliliters of the lipid droplet wash buffer. Centrifuge for two hours at 100, 000 times g and four degrees Celsius.
Following centrifugation, harvest the floating lipid droplet fraction from the top of the tube using a bent blunt cannula. Then, place the lipid droplet fraction in a centrifuge tube, overlay with lipid droplet wash buffer, and repeat the centrifugation step. Harvesting the lipid droplets from top of the gradient requires experience.
And is easier the bigger the fraction is. You can use fat cells for practice. Or you can feed your cells with oleate to increase the amount of lipid droplets.
Once again, harvest the floating lipid droplet fraction using a bent blunt cannula from the top of the tube. Transfer the lipid droplets to a new 1.5 milliliter microfuge tube, and add 500 microliters of lipid droplet wash buffer. Then, spin the sample for 20 minutes at 21, 000 times g and four degrees Celsius.
Remove the subjacent wash buffer using a gel loading pipette tip, and repeat the washing step three times. After the final removal of the washing buffer, mix five microliters of lipid droplet fractions with 10 microliters of NP 40 lysis buffer for determination of the protein content. Incubate the sample on ice for one hour to inactivate the virus if working with infectious material.
At this point, the lipid droplet fractions can be stored at minus 20 degrees Celsius. Next, mix the volume that corresponds to at least 35 micrograms of protein with 4x loading dye. Incubate the mixture on ice for one hour to inactivate the virus if working with infectious material.
Boil the sample at 95 degrees Celsius for five minutes. Then, perform SDS-PAGE followed by Coomassie staining to prepare the sample for liquid chromatogrophy couple to tandem mass spectronomy as described in the text protocol. To analyze the lipid droplet purity, dilute aliquots of lipid droplet fractions, one to two, with NP 40 lysis buffer.
Dilute input control fractions one to ten with the same buffer. Incubate the samples on ice for one hour to inactivate the virus if working with infectious material. Next, determine the protein level with a detergent compatible protein assay.
Then, mix equal amount of protein with 6x sample buffer, and incubate the samples on ice for one hour to inactivate the virus if working with infectious material. Finally, proceed with SDS-PAGE, and transfer the proteins to onto a nitrocellulose membrane by tank blotting at 80 volts for 90 minutes. Shown here is a representative western blot analysis of post-nuclear and lipid droplet fractions.
The western blot clearly shows an enrichment that the lipid droplet marker proteins perilipin two and perilipin three in lipid droplet fractions, and the depletion of markers of other cellular compartments. Post-nuclear and lipid droplet fractions can be additionally analyzed by Coomassie blue and silver staining. This heat map illustrates the number of peptides and percentage of protein coverage of proteins identified in lipid droplet fractions of the analyzed cells.
The final heat map depicts how HCV infection changes the lipid droplet per diem. Enriched lipid droplet-associated proteins are indicated in red, and depleted lipid droplet-associated proteins are indicated in blue. The main advantage of this method is that we mix the cells prior isolation and prior lipid droplet isolation, and thus we can minimize errors that can happen during the cell lysis and during the isolation steps.
