Feb 25, 2022
CIDC_S16_LC_MS_Celegans_Extraction_Protocol
- 1University of Georgia;
- 2Georgia Institute of Technology
Protocol Citation: Brianna M Garcia, Carter Asef 2022. CIDC_S16_LC_MS_Celegans_Extraction_Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bahjib4n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 13, 2019
Last Modified: February 25, 2022
Protocol Integer ID: 30987
Keywords: C. elegans, C elegans caenorhabditis elegans, LC-MSMS, LC-MS/MS, LC-MS, liquid chromatography, mass spectrometry, metabolomics, lipidomics
Abstract
A sample preparation protocol for lyophlized C. elegans samples to be analyzed via LC-MSMS
Materials
MATERIALS
Glass beads, acid-washed, 425-600 μm (30-40 U.S. sieve) Sigma AldrichCatalog #G8772-100G
AcetonitrileSigma AldrichCatalog #34998
Methanol HPLCFisher ScientificCatalog #9093-03
Eppendorf tubes 1.5 mL uncoloredEppendorf CentrifugeCatalog #022363204
2-PropanolFisher ScientificCatalog #A417-4
Water Optima™ LC/MS Grade Fisher ScientificCatalog #10728098
2.0mm zirconium oxide beads (Next Advance SKU ZROB20)Catalog #ZROB20
Homogenization
Homogenization
Samples are removed from -80 °C
(3) 2.0mm zirconium oxide beads and ~ 75 µL 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 00:03:00 .
Samples are now homogenized.
Extraction
Extraction
750 µL of 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 1.5 mL of solvent is transferred to the new 2.0mL Eppendorf tube.
Samples are vortexed for 00:01:00 and then extracted over night at -20C
Samples are placed in the centrifuge and spun at max speed (22100G) for 00:05:00
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 1.5 mL 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 00:30:00
2.0mL Eppendorfs are placed in the centrifuge and spun at max speed (22100G) for00:05:00
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
Sample drying/storage
Sample drying/storage
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 75 µL of 100% isopropanol for reverse phase and 80/20 methanol/water for HILIC.
Vortex for 1 minute, then centrifuge at max speed (22100G) for00:05:00
Transfer to LC-MS vial
After LC-MS analysis, samples can be stored at -80C until ready for IMS analysis