Code associated to the publication "Ozone-enabled fatty acid discovery reveals unexpected diversity in the human lipidome." by Jan Philipp Menzel, Reuben S. Young, Aurelie H. Benfield, Julia S. Scott, Puttandon Wongsomboon, Lukas Cudlman, Josef Cvacka, Lisa M. Butler, Sonia T. Henriques, Berwyck L.J. Poad and Stephen J. Blanksby, Nature Communications, 3940, 2023. https://www.nature.com/articles/s41467-023-39617-9
The code in this repository allows processing of LC-OzID-MS and LC-OzID-MS/MS files as well as mass spectrometry data from direct infusion ESI-MS as part of the workflow introduced in the associated publication. Usage of the code is explained in the Supplementary Information of the associated publication. Data associated to this publication (Raw data of LC-OzID-MS and LC-OzID-MS/MS files and Skyline transition lists. These transition lists require Skyline version 21.1.0.278.) are available via: https://researchdatafinder.qut.edu.au/display/n25697
IMPORTANT: Skyline (64-bit), version 21.1.0.278 was used for the analysis of the data in the associated publication. To use OzFAD, install Skyline, Visual Studio Code, python and download the zip file of this repository (<> Code - Download ZIP), unpack it and place it the folder OzFAD1.3 with all its contents in your main personal folder location. Please note for first-time installation of Skyline: Open the template.sky file, load the settings file 2021_09_15_SKYLINE_DE_NOVO_SETTINGS.skys from the folder location OzFAD1_black_box and save the settings; load an example transition list and an example dataset (associated to the Nat.Commun. paper) and if requested by Skyline, enter the lockmass (neg) as 556.2771 (LeuEnk) with a mass tolerance of 0.5. Load the report template skyline_report_vpw15.skyr from OzFAD1_black_box in the report interface of Skyline to export a first example report. If all this works, proceed to use the pipeline.
For installation and usage of python:
- Check, if multiple python versions exist (in cmd: "python --version")
- Uninstall all python version except the latest one (this one needs to be added to PATH, if unsure, uninstall and reinstall, adding python to PATH during installation).
- If required, open folder OzFAD1.3 in VS Code.
- Use pip to install packages, where required. To install pandas, type "pip install pandas" in terminal. To install PIL, type "pip install pillow". Note: If installation of packages with pip is problematic, anaconda may be easier to use compared to the original python.
- Run OzFAD1v3_GUI_7.py from VS Code or directly from the OzFAD1.3 folder.
NOTE: The latest versions of Skyline are currently not compatible with this workflow, work is in progress to ensure compatibility. Skyline Daily may be incompatible. To download version 21.1.0.278 of Skyline, go to the Skyline MS website > release installation page for your system architecture (here 64-bit) > follow link for unplugged installer > "I Agree" > "Archive" link below the "Download" link > Skyline (64-bit) 21.1.0.278. (try https://skyline.ms/labkey/_webdav/home/software/Skyline/%40files/installers/Skyline-64_21_1_0_278.zip)
If any problems with getting started with the OzFAD workflow remain or occur during data analysis, please contact me via ResearchGate, LinkedIn or E-mail. https://www.researchgate.net/publication/364795613_OzFAD_Ozone-enabled_fatty_acid_discovery_reveals_unexpected_diversity_in_the_human_lipidome >> Jan Philipp Menzel https://au.linkedin.com/in/jan-philipp-menzel-b09455b7
Latest updates to the workflow or this repository:
2025_08_29: Work is ongoing to fix compatibility issues with the latest Skyline version and the OzFAD workflow. Please use Skyline (64-bit), version 21.1.0.278 until further notice and whenever the workflow produces unexpected errors.
2023_09_21: Updated transition lists compatible with newer Skyline versions can be found in the folder Testing_files.
2023_05_10: The module for P value calculation now automatically writes mean values, standard deviations, fold changes, P values, t-test statistic values, degrees of freedom and confidence intervals (95%) into the output file.
2023_02_10: Speed improvement to algorithm for target list calculation. The algorithm demands less memory in case of long chromatographic gradients.
2023_01_09: Updated version OzFAD1.3 released:
- NEW: Graphical User Interface (GUI)
- NEW: All programs accessible via graphical user interface, which can be started either via a python program or an executable.
- NEW: Improved automated filtering after discovery step.
- NEW: Added files for testing of the workflow analysis steps (see folder Testing_files).
The files represent MCF7, replicate 1; FAME 37mix or Pooled Human Plasma NIST 1950 SRM.
- NOTES: If some steps of the workflow do not execute correctly after being started through OzFAD1v3_GUI_5.exe,
start the GUI from python program OzFAD1v3_GUI_6.py via Visual Studio Code (or other IDE).
To check, whether multiple python versions exist on the PC, in cmd type "where python".
If present, uninstall any old python versions as the workflow requires to automatically detect the python version under which the required packages are installed.
2022_11_16: The files in this repository are now in the folders in which they need to be locally for running the workflow. Paths to python do not need to be updated anymore, as long as python (any version of python3 should be fine) is installed correctly (Added to PATH during installation). To set up the workflow, simply download code as a zip file, unpack and copy OzFAD1.2 folder into your local home / personal folder; make sure that python with all relevant packages is installed, incl. IDE, as well as Skyline MS.
2022_11_14: The workflow is now compatible with latest Skyline versions. Two images explaining the steps of the workflow and the required folder structure for the files contained in this repository are added (Small changes to the versions inluded in the preprint).
2022_11_09: Speed improvements of initial precursor analysis and generation of target lists. The first batch file in the workflow may run significantly faster (< 90 sec) than outlined in the Supplementary Information of the preprint published on BioRxiv (> 6 min).
2022_11_02: Batch files now contain relative paths, only the path to the python version installed locally needs to be adjusted in each batch file, when setting up the workflow. Both python versions 3.9 and 3.11 (python.org) as well as other python versions should be compatible with the workflow.