OCT is a cryo-preservation agent that is incompatible with mass spectrometry analysis. Using this protocol, researchers can remove OCT from tissues and quantify sphingolipids with tandem liquid chromatography mass spectrometry. The preparation of tissue drying wicks, tissue washing, and tissue weighing will be demonstrated by April Boyd from Virginia Commonwealth University, and the lipid extraction will be demonstrated by Dr.Jeremy Allegood, director of the Virginia Commonwealth Metabolomics and Lipidomics Core.
To begin, clean surgical scissors by immersing them in methanol for five minutes, followed by wiping them with a clean laboratory grade tissue. Using powder-free gloves, twirl the end of a laboratory grade tissue to create a finely tapered 30 to 40 millimeter long wick. Then using the methanol cleaned scissors, cut the wick at the thick end and place it in a clean cardboard box.
Next, using the methanol cleaned scissors, cut four to five millimeters off the tip of a one milliliter pipette tip and place the tip back in the tip holder. To wash away the OCT from the tissues, add 10 milliliters of ice cold ultrapure deionized water to each tube containing human lung tissue and cap them tightly. Allow the tubes to incubate for 10 minutes.
Vigorously vortex each tube until all the tissue deposited on the walls of the tube or the tissue pellet is completely resuspended. Then centrifuge the tubes in a pre-chilled swinging bucket centrifuge at 4, 000 to 5, 000 times G for 10 minutes at four degrees Celsius. Once the centrifugation is complete, uncap the tubes on ice.
Carefully aspirate the wash solution, leaving approximately 0.5 milliliters of the supernatant in the tube, then recap the tube. After removing the supernatant from all tubes, wash the tissues twice more in the same manner. After the three OCT removal washes, use the prepared shortened pipette tip to add 500 microliters of ice cold PBS to the tube.
Using the same tip, gently resuspend the pellet and transfer all of the resuspended tissue into a pre-labeled and pre-weighed 1.5 milliliter centrifuge tube. Keep the tube on ice until all the tissue samples are transferred. Pellet the tissues by centrifuging the tubes in a pre-chilled centrifuge, then carefully aspirate as much of the PBS as possible without disturbing the pellet and place the tubes back on ice.
Using the tissue drying wicks prepared earlier, gently remove any remaining wash solution, then close the tube and place it on ice. To weigh the tubes, place one tube at a time in a recently calibrated tared and zeroed analytical balance and close the chamber door. Once the weight has stabilized, record it in an electronic spreadsheet.
After weighing, place the tube back on ice and add 300 microliters of ice cold PBS. Using a shortened pipette tip, carefully transfer all tissue into two milliliters of ice cold methanol in a pre-labeled screw top glass tube. Before lipid extraction, allow the tissues and mass spectrometry internal lipid standards to equilibrate to room temperature for 20 minutes, then vortex and immerse the internal standard solutions in an ultrasonic water bath until the lipid standards have fully dissolved.
Using a repeating pipette, add 250 picomoles of the internal mass spectrometry standards to each tube and lightly recap the tubes. To facilitate homogenization, adjust the volume of methanol to two milliliters, then triturate the tissue using a homogenizer by moving the homogenizer tip in a circular motion gently pressed against the tube wall. When clumps are no longer visible, recap the tube.
Immerse the tube at a 45 degree angle in an ultrasonic water bath for 5 to 10 seconds. Then under a fume hood, add chloroform and methanol to each tube to achieve a methanol to chloroform to water ratio of two to one to 0.1. Seal the tubes tightly and leave them on the benchtop until the end of the day.
Before leaving that evening, place the tightly capped tubes in a 48 degree Celsius water bath for an overnight incubation. On the following day, pellet any solvent and soluble debris by centrifugation. Then in a fume hood, carefully decant the supernatant into pre-labeled 13 into 100 millimeter borosilicate glass test tubes tilted at a 30 to 45 degree angle to facilitate the decanting.
After decanting the supernatant from all tubes, use a vacuum concentrator at maximum vacuum with an initial one-hour heating step at 40 degrees Celsius to evaporate all solvent, then add 500 microliters of methanol to each tube. To resuspend the dried lipid extracts, vortex the tube for 5 to 10 seconds, then immerse the tube in an ultrasonic water bath at a 45 degree angle with rotation for two minutes. Re-vortex the tube for five seconds and re-immerse in the ultrasonic water bath for an additional minute.
Remove any insoluble debris by centrifugation, then decant the supernatant into a pre-labeled autoinjector vial tilted at a 30 to 45 degree angle to facilitate the decanting. Cap the vials tightly before storing them at minus 80 degrees Celsius until analysis. Liquid chromatography electrospray ionization tandem mass spectrometry analysis following OCT removal confirms that various chain-length species of ceramides and monohexosyl-ceramides are significantly elevated in human lung adenocarcinoma tumors compared to normal adjacent uninvolved tissues.
Sphingolipid standards elute sequentially in the following order:d17:1 sphingosine, d17:1 sphingosine-1-phosphate, C12 lactosyl-ceramide, C12 monohexosyl-ceramide, C12 sphingomyelin, and C12 ceramide. For each of these sphingolipid species, using the gradient and instrumentation settings in the text, there is an approximate positive 0.15 minute shift in the retention time for every two carbon increase in chain-length. However, longer chain-length lipids such as C24 and C26 may have larger retention time shifts.
Furthermore, a double bond in the fatty acid often results in a negative 0.15 minute shift in retention time. Thus, C18:1 ceramide will have a retention time similar to C16 ceramide. Rather than relying almost exclusively on freshly collected tissues, this protocol expands the tissue sources that can be used for mass spectrometry analysis to include those cryogenically stored at biorepositories.
