Jun 12, 2025
Lipidomic analysis in HeLa cells for HexCer levels
Forked from Lipidomic analysis in HeLa cells
- Rosanne Wouters1,2,
- Igor Beletchi1,
- Chris Van den Haute3,4,2,
- Veerle Baekelandt3,2,
- Shaun Martin1,
- jan eggermont1,
- Peter Vangheluwe1,2
- 1Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD;
- 3Research Group for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven;
- 4Leuven Viral Vector Core, KU Leuven
Protocol Citation: Rosanne Wouters, Igor Beletchi, Chris Van den Haute, Veerle Baekelandt, Shaun Martin, jan eggermont, Peter Vangheluwe 2025. Lipidomic analysis in HeLa cells for HexCer levels . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxw13wv8j/v1
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: January 13, 2025
Last Modified: June 12, 2025
Protocol Integer ID: 118232
Keywords: lipidomic analysis in hela cell, hela cells for hexcer level, hela cell, lipidomic analysis, protocol for the lipidomics analysis, hexcer level, lipidomics analysis
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
C1 KU Leuven grant
Grant ID: C15/15/073
Fonds voor Wetenschappelijk Onderzoek (FWO) Flanders
Grant ID: S006617N SBO Neuro-Traffic
Michael J. Fox Foundation
Grant ID: MJFF-008610
Abstract
A protocol for the lipidomics analysis in HeLa cells for HexCer levels is described.
Troubleshooting
Lipidomics of Hexosylceramide in HeLa cells
Collect cells and homogenize cell pellets in 1 ml PBS (with calcium and magnesium).
To the homogenate, add sphingolipid internal standards.
Precipitate the protein with methanol and collect the supernatant.
Adjust the supernatant solution to 1:1 (v/v) methanol: water.
Lipid extraction: 700 μl of sample was mixed with 800 μl 1 N HCl:CH3OH 1:8 (v/v), 900 μl CHCl3 and 200 μg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma Aldrich).
Evaporate the organic fraction using a Savant Speedvac spd111v (Thermo Fisher Scientific) at room temperature.
Store the remaining lipid pellet at −20 °C under argon.
Just before mass spectrometry, reconstitute lipid pellets in running solution (CH3OH:CHCl3:NH4OH; 90:10:1.25; v/v/v)
Analyze phospholipid species by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a hybrid triple quadrupole/linear ion trap mass spectrometer (4000 QTRAP system; Applied Biosystems SCIEX) equipped with a TriVersa NanoMate (Advion Biosciences) robotic nanosource for automated sample injection and spraying.
Perform phospholipid profiling by (positive or negative) precursor ion or neutral loss scanning at a collision energy of 50 eV/45 eV, 35 eV, −35 eV, and −60 eV for precursor 184 (phosphatidylcholine (PC)), neutral loss 87 (phosphatidylserine (PS)) and precursor 241 (phosphatidylinositol (PI)), respectively.
Perform phospholipid quantification by multiple reactions monitoring (MRM), the transitions being based on the neutral losses or the typical product ions as described in step 8.
Relative quantification of phospholipids is obtained as ratio of analytes to their internal standard. The lipid data is normalized to nucleic acid concentration.