Mar 05, 2026
Lipidomics identifications and quantitation table generation using MS-DIAL (v4.9.221218)
- Jennifer Kyle1
- 1Pacific Northwest National Laboratory
- Pacific Northwest National Laboratory (PNNL)
Protocol Citation: Jennifer Kyle 2026. Lipidomics identifications and quantitation table generation using MS-DIAL (v4.9.221218). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld1jb7l5b/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: February 11, 2026
Last Modified: March 05, 2026
Protocol Integer ID: 243083
Keywords: Lipidomics, Identification, Pre-processing, raw data, lipidomics analysis of sample, based lipidomics analysis, lipidomics identification, lipid identification, mass spectrometry, lipid, protein extraction protocol, metabolite, liquid chromatography, analysis by liquid chromatography, sample preparation, mplex method, using m, raw file
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 indicates how to import Raw files, perform lipid identification in positive and negative ionization modes and export the intensity tables of datasets generated as follow:
- Sample preparation is performed using the protocol titled "Metabolites, Lipids, Protein extraction protocol (MPLEx)"
- Analysis by liquid chromatography coupled with mass spectrometry is performed following the protocol titled "Protocol for the LC-MS/MS-based lipidomics analysis of samples generated using the MPLEx method."
Materials
A computed equipped with MS-DIAL (v4.9.221218)
Troubleshooting
Loading Raw files in MS-DIAL
Note: this protocol is used on the samples generated using the protocol titled :
Open "MSDIAL.exe".
File -> New Project.
Click "Browse" Beside the "Project file path:" field (red circle below).
Under “Data type (MS/MS), check “Centroid data” (top red arrow).
Under “Target omics”, check “Lipidomics” (lower red arrow).
Under “Ion mode” select the ionization mode that aligns with the files to be processed (e.g., positive or negative ion mode)(red rectangle).
Click “Next”.
Click “Browse".
Change file type to “Raw file(*.raw)”.
Select raw files to be processed.
Optional – fill out remaining boxes with type of samples, batch, analytical order, etc.
Click "Next".
Parameter Selection for Data Processing
Select the following parameters per tab as shown (the parameters can also be uploaded using the “parameters_POS.med” file).
"Data collection" tab.
"Peak detection" tab.
"MS2Dec" tab.
"Analysis parameter setting" tab.
For “MSP file:” click “select” and check the following lipid subclasses for the "positive ionization".
Note: above are the subclasses selected for POS ionization.
NEG ionization selections used will be shown at the end of the document (post data export).
"Adduct" tab.
Note: See the end of the document for NEG mode selection
"Alignment" tab.
Note: see end of document for NEG mode selection
"Alignment" tab.
Select “Together with Alignment” and click “Finished.
Manual Examination of the Aligned Results
Double click on “alignmentresults…” (red arrow, lower left).
Double click on “alignmentresults…” (red arrow, lower left).
Select “Ref.match” and “MS2 acquired” (red circles).
Click “EIC aligned spot” to view aligned peaks.
Click “Show ion table” (red rectangle).
Once the alignment table opens (red box, below), it is recommended to sort by “Metabolite name” column then “RT(min)” column.
Manually validate the identifications by examining the MS/MS spectra.
To examine chromatograms to adjust and/or validate integration/selection, right-click by chromatograms and choose “Table viewer for curating each chromatogram” and/or “Aligned chromatogram viewer for simultaneous curations”.
Exporting Alignment Results
Save the data.
Click the “Export” tab.
Select “Alignment results".
Choose the export directory by clicking on “Browse”.
Choose the export directory by clicking on "Browse".
Check "Raw data matrix (Height).
Click "Export".
Differences in the Negative Ionization Mode
Parameter selection shown above is the same for both ionization modes with the following exceptions.
Identification tab.
"Adduct" tab.
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.