CIDC_S16_LC_MS_Celegans_Extraction_Protocol

Samples are removed from -80°C

(3) 2.0mm zirconium oxide beads and ~ 75µL volume of 0.5mm glass beads are added to each sample tube.

Samples are placed in Tissuelyser II using adapter trays chilled at -80C and homogenized at 1800rpm for 0h 3m 0s.

Samples are now homogenized.

750µLof 100% isopropanol is added to the 1.5mL Eppendorf tube containing the homogenized sample. The sample tube is lightly vortexed to create a suspension of homogenized sample and the resulting slurry is transferred to a new 2.0mL Eppendorf tube, leaving the beads behind in the original tube. This step is repeated so that a total of 1.5mL of solvent is transferred to the new 2.0mL Eppendorf tube.

Samples are vortexed for 0h 1m 0s and then extracted over night at -20C

Samples are placed in the centrifuge and spun at max speed (22100G) for 0h 5m 0s

Supernatant of each sample is transferred to a new 2.0 mL Eppendorf labeled for RP chromatography and dried down using steps 14 through 18

The second round of the sequential extraction is 1.5mL of 80/20 methanol/water per added directly to the pellet remaining after centrifugation.

Samples are shaken using the Fisher Scientific Isotemp High Speed Shaker at 1500rpm for 0h 30m 0s

2.0mL Eppendorfs are placed in the centrifuge and spun at max speed (22100G) for0h 5m 0s

Supernatant of each sample is transferred to a new 2.0 mL Eppendorf labeled for HILIC chromatography and dried down using steps 14 through 18

Pellets are dried for 1 hour and stored at -80°C

Samples are placed in a Labconco CentriVap concentrator and monitored until they have completely dried (roughly 4-5 hours)

Once dry, samples are stored at -80°C until they are to be run on the LC-MS instrument.

When preparing samples to run on LC-MS, reconstitute samples with 75µL of 100% isopropanol for reverse phase and 80/20 methanol/water for HILIC.

Vortex for 1 minute, then centrifuge at max speed (22100G) for0h 5m 0s

Transfer to LC-MS vial

After LC-MS analysis, samples can be stored at -80C until ready for IMS analysis