Jun 12, 2025
Lipidomic analysis in WM115 cells
Forked from Lipidomic analysis in HeLa cells
- Rosanne Wouters1,2,
- Igor Beletchi1,
- Chris Van den Haute3,4,2,
- Veerle Baekelandt3,4,2,
- 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;
- 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 WM115 cells . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dmn5vqe/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: 118148
Keywords: lipidomic analysis in wm115 cell, lipidomic analysis, protocol for the lipidomic analysis, wm115 cell
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
Michael J. Fox Foundation
Grant ID: MJFF-008610
Abstract
A protocol for the lipidomic analysis in WM115 cells
Troubleshooting
Lipidomic analysis in WM115 cells
Collect cells and homogenize cell pellets in 0.7 ml water with a handheld sonicator.
To the homogenate, add 0.8 ml HCl(1 M):CH3OH 1:8 (v/v), 0.9 ml CHCl3, 0.2 mg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma Aldrich), 3 μl of SPLASH‱ LIPIDOMIX‱ Mass Spec Standard ( Cat No: 330707, Avanti Polar Lipids), 3 μl of Ceramides and 3 μl of Hexosylceramides internal Standards (#5040167 and #5040398, AB SCIEX).
After vortexing and centrifugation, collect the lower organic fraction.
Evaporate the lower 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 the lipid pellets in 100% ethanol.
Analyze lipid species by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX).
Perform chromatographic separation on a XBridge amide column (150 mm × 4.6 mm, 3.5 μm; Waters) maintained at 35 °C using mobile phase A [1 mM ammonium acetate in water-acetonitrile 5:95 (v/v)] and mobile phase B [1 mM ammonium acetate in water-acetonitrile 50:50 (v/v)] in the following gradient: (0–6 min: 0 % B → 6 % B; 6–10 min: 6 % B → 25 % B; 10–11 min: 25 % B → 98 % B; 11–13 min: 98 % B → 100 % B; 13–19 min: 100 % B; 19–24 min: 0 % B) at a flow rate of 0.7 ml/min which was increased to 1.5 ml/min from 13 min onwards.
Measure phosphtidylserine, phosphtidylcholine and phosphtidylinositol in negative ion mode by fatty acyl fragment ions.
Perform phospholipid quantification by multiple reactions monitoring (MRM), the transitions being based on the neutral losses or the typical product ions.
The following instrument parameters are used: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = 5500 V and −4500 V; Temperature = 550 °C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = 60 V and −80 V; Entrance Potential = 10 V and −10 V; Collision Cell Exit Potential = 15 V and −15 V.
The following fatty acyl moieties were taken into account for the lipidomic analysis: 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:4, 22:5 and 22:6. Data Analysis:
Perform peak integration with the MultiQuant‱ software version 3.0.3. Lipid species signals were corrected for isotopic contributions (calculated with Python Molmass 2023.8.30;DOII:https://doi.org/10.5281/zenodo.7135495) and were quantified based on internal standard signals and adheres to the guidelines of the Lipidomics Standards Initiative (LSI) (level 2 type quantification as defined by the LSI).