Jun 10, 2026
Version 2
Untargeted Lipidomics - LC-MS Parameters of Q-Exactive - MetaboHUB Tours V.2
- Staff Members of PMAC1,2
- 1Plateforme de Métabolomique et d'Analyses Chimiques, US61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France;
- 2MetaboHUB-Tours, Tours, France
- MetaboHUB-Tours
External link: https://metabolomique.med.univ-tours.fr/
Protocol Citation: Staff Members of PMAC 2026. Untargeted Lipidomics - LC-MS Parameters of Q-Exactive - MetaboHUB Tours. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzeq88vx1/v2Version created by Jérémy Monteiro
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: June 10, 2026
Last Modified: June 10, 2026
Protocol Integer ID: 318842
Keywords: lipidomics, Q-Exactive, untargeted, LC-MS, lipidome coverage, analysis of lipid, lipid, system suitability cv of qc sample, ms² spectra, biological sample, present in biological sample, sample preparation, methods after sample preparation, qc sample, protein precipitation, cv of qc sample, untargeted lipidomic
Funders Acknowledgements:
Agence Nationale de la Recherche
Grant ID: ANR-11-INBS-0010 MetaboHUB
Agence Nationale de la recherche au titre de France 2030
Grant ID: ANR-21-ESRE-0035
Abstract
I. PURPOSE
Identification and relative quantification (based on intensity) analysis of lipids present in biological sample for lipidome coverage.
II. MATERIAL & METHODS
After sample preparation which consisted in a protein precipitation, samples are analyzed by ES-LC-HRMS (Q-Orbitrap) by 2 modalities: C18 in both positive and negative mode.
III. DATA TREATMENT
Data are analyzed using Workflow4Metabolomics open-source software that allows to detect all features.
Features are identified with LipidSearch5.0 software, based on the m/z and the MS² spectra.
IV. QUALITY CONTROL
Checking the system suitability
CV of QC samples (injected/10 injections) < 30 %
Guidelines
Metabolomics, and so lipidomics, are the “omics” sciences studying metabolites and can meet the need for a deeper insight into biological data. It offers a real-time snapshot of a metabolic state and so reflect immediate physiological changes.
By revealing metabolic & lipidic profiles, they allow for precise diagnosis, tailored treatments, and proactive health management, ensuring highly personalized and effective interventions.
(Nordström, A., Lewensohn, R. Metabolomics: Moving to the Clinic. J Neuroimmune Pharmacol 5, 4–17 (2010). https://doi.org/10.1007/s11481-009-9156-4 )
Untargeted lipidomicis analyses were performed on an UPLC Ultimate WPS-3000 system (Dionex, Germany) coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a HESI source (head electrospray ionisation). Xcalibur 2.2 software (Thermo Fisher Scientific, Bremen, Germany) controlled the system.
For exploratory analyses, features were considered for further analysis only if they were detected in all injections and their CV were less than 30 % after signal normalization. The identification of metabolites was performed using LipidSerch5.0, based on MS² spectra and validated with retention time of an external sample well-characterized by the laboratory.
Materials
Equipment
Q-Exactive
NAME
Mass Spectrometer
TYPE
ThermoScientific
BRAND
Protocol materials
formiate ammoniumVWR International (Avantor)
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Isopropanol HyperSolv CHROMANORMVWR International (Avantor)
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
Aluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)
Calibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher
Calibration Solution Pierce ESI Negative IonThermo Fisher
Safety warnings
All solutions must be carried out under a fume hood, equipped with traditional PPE: lab coat, goggles, and gloves.
Ethics statement
All samples were obtained in accordance with French legislation (Loi Jardé). In French hospitals with a research mission, the use of sample leftovers for research purposes is based on the principle of no opposition. According to Loi Jardé, no requirement of Ethical Committee or Institutional Review Board was required because no new sampling was required. This law also stipulates that patients must be fully informed of this information/practice and patients must have the possibility, at any time, of objecting to the use of their samples in research. The analyses were always performed in accordance with confidentiality rules. Data were coded without mention of first and last names, and the results were produced in a way that does not allow patients to be identified. Patients did not invoke their right to have a right of access, rectification, portability and limitation of the processing of data and/or biological samples.
Reversed-Phase Analysis (BEH-C18, +/-)
Phase A :
- 200 mL + 4.0 mL * [Number of injections]
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) /Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) (5:3:2) + 10 millimolar (mM) formiate ammoniumVWR International (Avantor)
- Stir vigorously during 00:01:00
- Ultrasound during 00:10:00 to remove air bubble
Phase B:
- 200 mL + 4.0 mL * [Number of injections]
- Isopropanol HyperSolv CHROMANORMVWR International (Avantor) /
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (90:9:1) + 10 millimolar (mM) formiate ammoniumVWR International (Avantor)
- Stir vigorously during 00:01:00
- Ultrasound during 00:10:00 to remove air bubble
Chromatographic System
Equipment
Ultimate 3000
NAME
UHPLC
TYPE
Dionex Softron
BRAND
SRD-3400
SKU
Pump, Column Compartment, AutoSampler
SPECIFICATIONS
Install the column in the oven
Equipment
ACQUITY UPLC BEH C18 (2.1x100x1.7)
NAME
Column
TYPE
Acquity
BRAND
Adjust the temperature (60 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.4 mL/min at 2%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0 | 0.4 | 2 | |
| 0 | 0.4 | 2 | |
| 2 | 0.4 | 2 | |
| 6 | 0.4 | 30 | |
| 9 | 0.4 | 75 | |
| 9.5 | 0.4 | 100 | |
| 11.8 | 0.4 | 100 | |
| 12 | 0.4 | 2 | |
| 13.7 | 0.4 | 2 |
Mass Spectrometer:
Equipment
Q-Exactive
NAME
Mass Spectrometer
TYPE
ThermoScientific
BRAND
HESI Source
SPECIFICATIONS
MS Parameters :
- Scan Range: 250-1500 m/z
- Resolution MS1: 70,000
- Resolution MS2: 15,000
- Polarity: POS or NEG
- Microscans: 1
- Lock mass: None
- AGC Target: 1E6
- Maximum Inject Time: 200
- Sheath Gas Flow Rate: 45
- Aux Gas Flow rate: 10
- Sweep Gas flow Rate: 1
- Spray Voltage (kV): +/-3.5
- Capillary Temperature (°C): 250
- S-lens RF level: 25
- Aux gas Heater temperature (°C): 350
Cleaning the source:
- Set source temperature to 50 °C
- Let it "cool" 01:00:00
- Unplug the Ion MAx API source with HESI probe attached
- Unscrewed the Sweep Cone and the capillary
- Immediately plug another cleaned capillary
- Clean the sweep cone with wet Aluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)
- Rinse thoroughly with Water milliQMerck MilliporeSigma (Sigma-Aldrich)
- In a glass beaker, cover completely the Sweep Cone and the capillary with Methanol HiPerSolv CHROMANORMVWR International (Avantor)
- Ultrasound during 00:10:00
- Let it dry
- Replace the cone and the Ion Max API source with HESI probe
- Set source temperature back to normal
Reinstall the source & allow it to return to analysis conditions (described above)
Calibration (direct infusion):
- POS: Calibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher
- NEG: Calibration Solution Pierce ESI Negative IonThermo Fisher
- RMS < 0.4/0.4 in both ionisation mode
Once calibrated, sequences can be run
Typical Sequences:
| Equilibrium(=sample) * 10 | 5 | |
| Blank | 5 | |
| Performance Evaluation * 3 | 5 | |
| Blank | 5 | |
| Blank Solvent #1 | 5 | |
| Blank Solvent #2 | 5 | |
| Blank Extraction #1 | 5 | |
| Blank Extraction #2 | 5 | |
| Sample QC | 5 | |
| Sample *10 | 5 | |
| Sample QC | 5 | |
| Continue the sequence, alternating between samples and a QC | 5 | |
| Last Sample QC | 5 | |
| QC in MS² | 5 | |
| Blank Solvent * 3 | 5 |
Metabolite Identification :
- Lipids are identified based on MS²
Software
LipidSearch
NAME
ThermoScientific
DEVELOPER
- The list of m/z and RT, from the identified lipids, are compared with a "reference"one, from a well-characterized sample (mix of multiple lyophilized biological samples)
Features detection:
- Raw files are converted
Software
MSconvert
NAME
Chambers, M., Maclean, B., Burke, R. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30, 91
DEVELOPER
- Using Workflow4Metabolomics :
- The data were processed using the XCMS package in R.
- Molecules were then identified according to the following criteria:
-> Retention time delta with reference: 20 seconds
-> Experimental vs. theoretical mass delta: 10 ppm
- Once the Excel data table with the detectedmetabolites was obtained, the data were filtered (following steps)
Software
Workflow4Metabolomics
NAME
doi: 10.1093/bioinformatics/btu813
DEVELOPER
REPOSITORY
SOURCE LINK
- Data Curation = Signal Normalization & Lipids Exclusion :
- Normalization of lipids are normalized as to the total area of detected lipids
- Calculation of the coefficient of variation (CV) for each lipid in the QCs (analytical variability) and in the samples (biological variability)
- Removal of lipids with analytical variability > biological variability
- Removal of lipids with analytical variability > 30%
- Signal Normalization and Metabolites Exclusion are repetead until there is no further exclusion
Analysis Validation: the analyses were validated by observing the distribution of QCs among the samples using Principal Component Analysis (PCA). For all PCAs, the data were log-transformed and underwent UV scaling normalization.
Software
SIMCA
NAME
Sartorius
DEVELOPER
Fusion of lipids = sorting redundancies:
- Only the best modality is kept for 1 lipid, based on RT > Dead Volume (0-1 min) and/or lower CV(lipid) on QCs
- "Analysis Validation" is proceed
- Assignment of CHEBI identifiers des for those referenced in the CHEBI database
Software
CHEBI
NAME
Malik, A., Arsalan, M., Moreno, C., Mosquera, J., Félix, E., Kizilören, T., Muthukrishnan, V., Zdrazil, B., Leach, A. R., and O'
DEVELOPER
SOURCE LINK
- Specific identifiers for the “Homo sapiens - Recon 2_2” metabolic network are also available, allowing them to be viewed via MetExplore3
Software
MetExplore
NAME
Cottret Ludovic, Frainay Clément, Chazalviel Maxime, Cabanettes Floréal, Gloaguen Yoann, Camenen Etienne, Merlet Benjamin, Porta
DEVELOPER
OpenScience
"Open science has the potential of making the scientific process more transparent, inclusive and democratic. Open science:
- increases scientific collaborations and sharing of information for the benefits of science and society;
- makes multilingual scientific knowledge openly available, accessible and reusable for everyone; and
- opens the processes of scientific knowledge creation, evaluation and communication to societal actors beyond the traditional scientific community.
Our interconnected world needs open science to help solve complex social, environmental, and economic challenges and achieve the Sustainable Development Goals."
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Software
MetaboLights
NAME
Ozgur Yurekten, Thomas Payne, Noemi Tejera, Felix Xavier Amaladoss, Callum Martin, Mark Williams, Claire O’Donovan. MetaboLights
DEVELOPER
SOURCE LINK