Jun 15, 2026

Lipidomics Data Analysis and Background Normalization

DOI
https://dx.doi.org/10.17504/protocols.io.ewov11o27vr2/v1
  • Elisabeth Holzer1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov11o27vr2/v1
Protocol Citation: Elisabeth Holzer 2026. Lipidomics Data Analysis and Background Normalization. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11o27vr2/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
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Created: August 06, 2025
Last Modified: June 15, 2026
Protocol  Integer ID: 224293
Keywords: ASAPCRN, lipidomics data analysis, raw lipidomics data, lipid class grouping, background normalization, background normalization this protocol, pmol, normalization, background subtraction, molar percentage
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol describes how to process and analyze raw lipidomics data (in pmol), calculate molar percentages (%mol), and perform background subtraction using Microsoft Excel. The procedure includes replicates averaging, normalization, and lipid class grouping.
Procedure:
Open the raw lipidomics data in Microsoft Excel.
Replace missing values in the raw data with 0 to avoid calculation errors.
Average the replicates of the MOCK control:
For each lipid species, calculate the mean value across the three biological replicates.
Subtract the background (MOCK control):
Subtract the averaged MOCK control values from each replicate of the ATG9A vesicle samples for each lipid species.
If the result is negative, set the value to 0. The background-corrected values were then used to calculate the final %mol values.


Calculate the total lipid amount:
For each condition (after background subtraction), sum all lipid species to obtain the total lipid content.
Calculate molar percentage (%mol):
Use the formula: %mol = (individual lipid species / total lipids of that condition) × 100
To obtain the final lipid composition, calculate the mean molar percentage across all replicates (for each group of lipids).
Group lipids into categories:

  • Cholesterol
  • PC: Includes PC and LPC
  • PE: Includes PE and LPE
  • PI
  • PS
  • SM
  • OTHERS: Includes CE, Cer, DAG, GlcCer, LacCer, PG, and TAG
Generate summary tables or graphs for visualization and statistical analysis as needed.