Mar 06, 2026
Protocol for the LC-MS/MS-based lipidomics assay of samples generated using the MPLEx method
- Josie Eder1,
- Jennifer Kyle1
- 1Pacific Northwest National Laboratory
- Pacific Northwest National Laboratory (PNNL)
Protocol Citation: Josie Eder, Jennifer Kyle 2026. Protocol for the LC-MS/MS-based lipidomics assay of samples generated using the MPLEx method . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx85bwv8j/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 30, 2026
Last Modified: March 06, 2026
Protocol Integer ID: 241808
Keywords: Lipidomics, LC-MS/MS, MPLEx, HubMAP, total lipid extract, hubmap lung sample, based lipidomic, tandem mass spectrometry, liquid chromatography, mplex method, mplex protocol
Funders Acknowledgements:
NIH - Biorepository for Investigation of Neonatal Diseases of Lung-Normal (BRINDL-NL)
Grant ID: U01HL122700
NIH - Biorepository for INvestigation of Diseases of the Lung (BRINDL) - Phase II
Grant ID: U01HL148861
NIH - The Human Lung BioMolecular Multi-Scale Atlas Program (HuBMAP-Lung)
Grant ID: U54HL165443
NIH - Research Center for Spatiotemporal Lung Imaging and Omics
Grant ID: U01HL148860
Abstract
This protocol describes how Total Lipid Extracts (TLEs) obtained from the MPLEx protocol are analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The details provided here indicate what was performed for the HubMAP Lung samples.
Materials
For the MPLEx procedure
Chloroform
Methanol
MilliQ water
Vortex
Speedvac
For the LC-MS/MS
Mobile phase A (MPA): 10 mM ammonium acetate in 60% Milli-Q H2O and 40% acetonitrile
Mobile phase A (MPA): 10 mM ammonium acetate in 60% Milli-Q Hl and 10% acetonitrile
Waters CSH 1.7 µm, 3 mm x 150 mm length chromatography column
Vanguard pre-column with the same packing material as the main column.
Waters H-Class liquid chromatography system
Thermo Scientific Lumos Mass spectrometer
Troubleshooting
Lipid Sample Preparation
Lipid are extracted using a modified Folch extraction (Folch 1957; Nakayasu, et al., 2016) termed MPLEx.
The protocol named: "Metabolites, Lipids, Protein extraction protocol (MPLEx)" can be followed to do this.
The lower organic layer was removed, dried in vacuo then stored at -20 ˚C in chloroform:methanol (2:1, v/v) until MS analysis.
Just before analysis, Total Lipid Extracts were reconstituted in 100 µL of 10% chloroform and 90% methanol. 45 µl were transferred to two separate vials, one to be used for positive ionization mode and one for negative ionization mode analysis.
Lipidomics Mass Spectrometry
5 µl is injected in the liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system, comprising a Waters H-Class liquid chromatography system interfaced with a Thermo Lumos mass spectrometer.
Note: Vialed samples can be conserved for up to 3 months in a -20 ˚C freezer
The ESI source parameters are set as follows: spray voltage 3.5 or 3.4 kV for positive and negative modes respectively; capillary temperature 350 °C; S lens RF level 30 arbitrary units; aux gas heater temperature 350 °C; sheath, auxiliary, and sweep gas flows of 50, 10, and 1, respectively.
Full MS scans were acquired at a resolving power of 120,000 FWHM at m/z 200 with the scanning range of m/z 200–1800. The data dependent acquisition (dd-MS2) parameters used to obtain product ion spectra are as follows: time limit of 1s with scans alternating between CID and HCD, isolation width of 2 m/z units, default charge state of 1, activation Q value of 0.18 for CID, HCD resolving power of 7,500 FWHM at m/z 200, normalized collision energies for CID of 38 with detection in the ion trap and stepped collision energy of 25, 30, and 35 for HCD with detection in the orbitrap.
The mass spec method was set for a 25 minute acquisition for both polarities.
Lipidomics Liquid Chromatography
The column used was a Waters CSH 1.7 µm, 3 mm x 150 mm length paired with a Vanguard pre-column with the same packing material held at a temperature of 50 °C. An injection volume of 10 uL was used for both polarities.
All solvents and additives used were LC-MS grade. Mobile phase A (MPA) consisted of 10 mM ammonium acetate in 60% Milli-Q H2O and 40% acetonitrile. Mobile phase B (MPB) consisted of 10mM ammonium acetate in 90% isopropanol and 10% acetonitrile. The same solvents were used for both modes with the full amount needed in each mode being mixed beforehand to reduce lipid retention time shifts.
The following gradient was used for both polarities. Note that minutes 25 to 29 of the gradient are the wash and equilibration portion and are not acquired by the Lumos.
| Time (min) | Flowrate (mL/min) | % MPA | % MPB | |
| 0 | 0.3 | 60 | 40 | |
| 1 | 0.3 | 38 | 62 | |
| 4 | 0.3 | 34 | 66 | |
| 9 | 0.3 | 22 | 78 | |
| 11 | 0.3 | 13 | 87 | |
| 15 | 0.3 | 1 | 99 | |
| 21 | 0.3 | 1 | 99 | |
| 21.1 | 0.3 | 60 | 40 | |
| 21.6 | 0.3 | 1 | 99 | |
| 21.7 | 0.3 | 60 | 40 | |
| 25 | 0.3 | 60 | 40 | |
| 29 | 0.3 | 60 | 40 |
QA and QC
Polysiloxane in positive mode and palmitic acid in negative mode are monitored for its part per million (ppm) mass shift, and iQC chromatographs across a sample set are superimposed to check for retention time shifts. If the ppm error is higher than 5 ppm or changes 5 or more ppm over a sample set, the sequence is aborted and a mass calibration is rerun. A drift in elution time of 5 seconds or more also requires the sequence to be stopped and indicates an LC error. Mobile phase solvents are changed between experiment sets (i.e., not during a sample set) to reduce drifting retention times. The retention time, ppm error, and intensity for polysiloxane and PC 16:0/18:1 in positive mode and palmitic acid and PS 18:0/18:1 in negative mode are tracked to monitor the instrument’s performance long term.
Prior to MS analysis, 20 µL of total lipid extract taken from each reconstituted sample was combined to form a Pooled QC (pQC). The following sequence made up quality assurance and quality control blocks: iBlank, IQC of 0.25 mg/mL BTLE, iBlank, pQC, iBlank.
Data Analysis
The data analysis is performed using the protocol titled:
Acknowledgements
This work was supported by the National Heart, Lung, and Blood Institute (NHLBI) Molecular Atlas of Lung Development Program Human Tissue Core (LungMAP HTC) and LungMAP BioRepository for INvestigation of Diseases of the Lung (BRINDL) through grants U01HL122700 and U01HL148861 (to GS Pryhuber), and U01HL148860 (G. Clair) and by the NIH Common Fund grant U54HL165443 (to GS Pryhuber with Co-Investigators G Clair, and C Anderton). Part of this work was performed in the Environmental Molecular Science Laboratory, a U.S. Department of Energy (DOE) national scientific user facility at Pacific Northwest National Laboratory (PNNL). Battelle operates PNNL for the DOE under contract DE-AC05-76RLO01830. The opinions expressed in this article are the authors’ own and do not reflect the view of the NIH, the Department of Health and Human Services, or the U.S. government. We are very grateful for the generosity of the donor families and honor their loss.