Feb 25, 2026
Lipid Trap Mass Spectrometry (LTMS) protocol - Eggert lab
- Andrea Paquola1,2,
- Cagakan Ozbalci1,
- Ulrike Eggert1,2
- 1Randall Centre for Cell and Molecular Biophysics, King’s College London, New Hunt’s House, Guy’s Campus, SE1 1UL, UK;
- 2Department of Chemistry, King’s College London, London, UK, SE1 1UL, UK
- Andrea Paquola
Protocol Citation: Andrea Paquola, Cagakan Ozbalci, Ulrike Eggert 2026. Lipid Trap Mass Spectrometry (LTMS) protocol - Eggert lab. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv55md4v1b/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
Created: February 16, 2026
Last Modified: February 25, 2026
Protocol Integer ID: 243405
Keywords: lipid trap mass spectrometry, trap mass spectrometry, eggert lab the lipid, ltm, lipid interactions in mammalian cell, lipid interaction, lipid
Funders Acknowledgements:
Wellcome Trust
Grant ID: 110060/Z/15/Z
BBSRC
Grant ID: BB/X000605/1
Abstract
The Lipid-Trap Mass Spectrometry (LTMS) is a method to study protein-lipid interactions in mammalian cells. The detailed step-by-step procedure is described in this protocol.
Troubleshooting
Harvest cells
Plate 1 × 10⁶ HeLa cells stably expressing either GFP-tagged protein of interest or MyrPalm-GFP (negative control) per 10cm dish. Prepare three independent biological replicates per condition.
The day after transfer dishes onto ice for 5 min. This allows to slow cellular metabolism and preserve transient protein-lipid interactions.
Aspirate growth medium and wash with 10 mL ice-cold DPBS.
Lyse cells
Filter and add 1 mL ice-cold PIPES 20mM buffer, pH=6.8 (you will have to adjust the pH with NaOH 1M) supplemented with 1xProt inhibitor PMSF 1 mM (Roche).
Harvest cells by gently scraping them using a cell scraper.
Transfer lysates to pre-chilled 1.5-mL Eppendorf tubes kept on ice.
Sonicate using probe sonicator. Sonication parameters: 20 s ON / 30 s OFF, total of 3 min, at 30 kHz, keeping samples on ice. This disrupts cells mechanically while maintaining protein-lipid complexes.
Centrifuge at 3,500×g for 10 min at 4°C to remove debris.
Save 20 μL of each lysate as input control for Western blotting, allowing verification of protein expression and recovery.
Wash beads
Wash GFP magnetic agarose beads (Chromotek) twice with 500 μL of ice-cold PIPES buffer to remove preservatives and equilibrate.
Bind proteins
Add 40 μL of equilibrated beads per 1 mL of sample.
Incubate for 1 h at 4 °C on a tube rotator to facilitate efficient binding.
Wash beads
Place Eppendorf tubes on a magnetic rack and remove supernatant.
Wash beads twice with 500 μL of ice-cold PIPES buffer to reduce nonspecific interactions while preserving protein-lipid complexes.
Downstream options
For lipid analysis → proceed to "Extraction lipids" Section.
For protein analysis → resuspend beads in 20 μL of 3× sample buffer with DTT, boil 10 min at 95 °C, and perform Western blot.
Extraction lipids
Transfer beads (suspended in 500 μL of ice-cold PIPES) from Eppendorf to glass Wheaton tubes (Fisher Scientific Ltd). Place tubes on a magnetic rack (vertically) and remove any remaining aqueous phase carefully to avoid loss of beads.
Neutral lipids (Folch Method1)
Add 250 μL CHCl₃/MeOH (2:1, v/v). Vortex twice for 60 s to solubilize neutral lipids efficiently.
Carefully transfer the organic phase into chloroform-resistant microcentrifuge tubes using a glass Pasteur pipette. Take care to avoid transferring any beads.
Evaporate organic phases at 37 °C under constant N₂ flow to prevent oxidation.
Resuspend dried lipids in appropriate LC–MS loading buffer.
Phosphoinositide extraction2 (modified Folch method) and derivatization3
Add 726 μL CHCl₃/MeOH/1 M HCl (40:80:1, v/v/v) to beads in glass tubes and vortex 15 min.
Add 720 μL CHCl₃, vortex 5 min, then 354 μL 1 M HCl, vortex 2 min.
Centrifuge 1,000 × g for 5 min.
Transfer the lower (organic) phase containing phosphoinositides to a fresh glass tube.
Add 726 μL CHCl₃/MeOH/1 M HCl to the glass tube containing the acqueous phase from step 22, vortex 10 s, centrifuge 1,000 × g for 5 min.
Transfer the lower (organic) phase and pool it with the first organic extract (step 25).
Evaporate pooled organic phases at 37 °C under constant N₂ flow to prevent oxidation.
Resuspend dried phosphoinositide extracts in 90 μL MeOH/CH₂Cl₂ (4:5, v/v). Perform derivatization in fume hood.
Add 10 μL TMS-diazomethane (2 M in hexane) (Sigma-Aldrich). The reagent is toxic. Follow all safety protocols.
Incubate 30 min at room temperature for methylation.
Add 20 μL glacial acetic acid (quenching).
Keep a beaker of acetic acid in fume hood to neutralize volatiles.
Evaporate mixture at 37 °C under constant N₂ and resuspend in LC–MS loading buffer.
Data processing, export and statistical preprocessing
Process raw data and extract features using mass spectrometer-specific software.
Remove features originating from bead-only controls and solvent blanks.
Assign samples to experimental groups: MyrPalm-GFP (control) and GFP-protein of interest (PoI) pull-downs.
Group biological replicates for each condition.
Retain only features detected in ≥ 80% of samples in at least one group.
Statistical comparison: MyrPalm-GFP vs. protein pull-down
Perform one-way ANOVA using mass spectrometer processing software with a significance threshold: p < 0.05.
Apply a Tukey HSD post-hoc test.
Perform pairwise comparisons of each GFP-protein pull-down against MyrPalm-GFP (control).
Manual verification and quantitative re-exportation
Based on the statistical comparison output, re-inspect all statistically significant features in mass spectrometer processing software.
Export quantitative feature tables as CSV files containing: m/z, retention time and peak area.
Multiple-testing correction and final significance testing
Import the CSV files into GraphPad Prism (GraphPad Software).
Perform multiple testing correction using: false discovery rate (FDR), two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, Q = 5%.
Retain only features passing the FDR threshold.
Targeted analysis for phosphoinositides
Generate extracted ion chromatograms (EICs) for lipids with known m/z and retention time (e.g. derivatized phosphoinositides) using mass spectrometer processing software.
Export EIC peak areas.
Analyze EIC areas in GraphPad Prism using: two-sided t-tests, FDR correction.
Selection of features for MS/MS
Select features for MS2 analysis only if they: show statistically significant changes between MyrPalm-GFP and GFP-PoI pull-downs, and pass manual inspection of peak shape, alignment and signal quality.
MS/MS acquisition and lipid annotation
Acquire MS2 spectra for all selected features.
Perform lipid annotation by matching MS2 fragment ions using the following databases/tools: LIPID MAPS, MS-DIAL and/or MS-FINDER.
Use fragment ion patterns to assign lipid classes and molecular species.
Use of total HeLa lipid extracts to support identification
If MS2 spectra are difficult to interpret due to background noise, analyze the same features in total HeLa lipid extracts. Use the higher abundance in total extracts to confirm fragment patterns and increase confidence in lipid assignments.
Criteria for lipid assignment
Assign lipids based on a combination of MS2 fragment matching, accurate mass (≤ 5 ppm deviation), retention time and adduct formation patterns.
Protocol references
1. Folch, J., Lees, M. & Sloane Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226, 497-509 (1957).
2. Mucksch, F. et al. Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes. Sci Rep 9, 17661 (2019).
3. Traynor-Kaplan, A. et al. Fatty-acyl chain profiles of cellular phosphoinositides. Biochim Biophys Acta Mol Cell Biol Lipids1862, 513-522 (2017).