CyanoPeptideMatching (CPM) Python Version

This project provides a comprehensive analytical workflow for analyzing LC-MS/MS mass spectrometry fies (mzML format) for the rapid identification and classification of cyanobacterial secondary metabolites. CPM utilizes diagnostic-ion-guided MS/MS querying for specific cyanopeptide classes, precursor-level MS1 consolidation, adduct-feature relationship mapping, and database-assisted matching to CyanoMetDB. The workflow employs the MassQL query language to perform flexible, reproducible searches across MS2 spectra based on class-specific diagnostic ions, mass tolerances, and retention time windows. CPM supports relative trend analysis by extracting MS1 chromatographic signal directly from the user-provided LC–MS/MS datasets within the workflow. Extracted ion chromatograms (EICs) are generated for matched precursor features, and area under the curve (AUC) values are computed to provide relative abundance estimates. A user-defined reference compound enables normalization of MS1 AUC values across samples, facilitating relative comparison of feature abundances while maintaining a streamlined analysis pipeline.

• Technologies used * Proteowizard (converting raw mass spectrometry files to .mzML) * Python (Python 3.13)

Getting Started

Linux/macOS

Click to expand for information on how to set up CPM!

Open terminal

Install micromamba once

cd ~
curl -Ls https://micro.mamba.pm/install.sh | bash
source ~/.bashrc

Create environment

micromamba create -n cpm python=3.13 pip -c conda-forge -y

Activate it

micromamba activate cpm

Check Python version (should be 3.13.x)

python --version

Upgrade install tools

pip install --upgrade pip setuptools wheel

Install CPM package

pip install --no-cache-dir --force-reinstall
git+https://github.com/sheffera01/CyanopeptideMatchingPythonVersion.git@package

For Future Logins

source ~/.bashrc
micromamba activate cpm

For newest version pulling from GitHub:

pip install --no-cache-dir --force-reinstall
git+https://github.com/sheffera01/CyanopeptideMatchingPythonVersion.git@package

To run:

cpm
--class-tag MC \
--files /path_to_file/filename.mzML \
/path_to_file/filename2.mzML \
--metadata /path_to__metadata/metadata.csv \
--output-root /path_to_result_output/results \
--blank-filter \
--batch-correct

Use cpm --help for usage information

Options:

--class tag: MC, AP, AB, AR, MG, or ALL
--blank-filter: optional. Remove if not needed
--batch-correct: optional. Remove if not needed.

PC

Click to expand for information on how to set up CPM!

Open PowerShell

Install micromamba once

1. cd $HOME
2. Invoke-WebRequest -Uri https://micro.mamba.pm/api/micromamba/win-64/latest -OutFile micromamba.tar.bz2
3. tar xf micromamba.tar.bz2
4. .\Library\bin\micromamba.exe shell init -s powershell -r "$HOME\micromamba"

Close PowerShell and reopen it.

Create environment

micromamba create -n cpm python=3.13 pip -c conda-forge -y

Activate it

micromamba activate cpm

Check Python version (should be 3.13.x)

python --version

Upgrade install tools

pip install --upgrade pip setuptools wheel

Install CPM package

pip install --no-cache-dir --force-reinstall "git+https://github.com/sheffera01/CyanopeptideMatchingPythonVersion.git@package"

#-----------------------------------------------------------------------------------------------

For Future Logins

Open PowerShell and run: micromamba activate cpm

For newest version pulling from GitHub:

pip install --no-cache-dir --force-reinstall "git+https://github.com/sheffera01/CyanopeptideMatchingPythonVersion.git@package"

#-----------------------------------------------------------------------------------------------

To run:

cpm --class-tag MC --files "C:\path_to_file\filename.mzML" "C:\path_to_file\filename2.mzML" --metadata "C:\path_to_metadata\metadata.csv" --output-root "C:\path_to_output\results" --blank-filter --batch-correct \

Use cpm --help for usage information

Options:

--class tag: MC, AP, AB, AR, MG, or ALL
--blank-filter: optional. Remove if not needed
--batch-correct: optional. Remove if not needed. \

Help Function in CPM

Use cpm --help for usage information in the CPM package!

Prerequisites

#%pip install pandas (2.3.3) matplotlib (3.10.8) numpy (2.2.6) seaborn (0.13.2) networkx (3.4.2) massql (2025.12.10) openpyxl (3.1.5) pyteomics (4.7.5) lxml (6.0.2)
need to be running also in massql_env (Python 3.13.X)

Areas for users to edit specific parameters

Adding to ion search lists

• cpm.py --> edit Ion Lists AND Ion label dictionary

Blank Ratio Threshold

• cpm.py --> blank_ratio_threshold

Tolerance matching to CyanoMetDB

• cyanometdb_match.py --> edit tol_da: float = __ AND
• cpm.py --> edit tol_da: float = 0.05,

Number of Diagnostic Product Ions Needed for matches

• cyanometdb_match.py --> "n_diagnostic"] >= 2

Adduct additions

• adduct_finder.py --> add into default_deltas

Adjusting merged summary

• summary_builder.py --> merge_tol_mz and merge_tol_rt edit

MS1 AUC

• summary_builder.py --> tol_mz and rt_pad edit

MS/MS diagnostic ion tolerance fragment

• massql_utils.py --> tol_mz

Acknowledgements

• We are grateful for support from the National Institute of Environmental Health Sciences (NIEHS) of the NIH under award numbers 5P01ES028939-02 and R21ES033758 (M.J.B.) and the National Science Foundation (NSF) under award number OCE-1840715, T32 GM140223 Pharmacological Sciences Training Program (S. L. H.), the National Institute of Health (NIH) F31 1F31ES036421-01 (L.N.H.), the National Institute of Health (NIH) F31 1F31AI186432-01 (K.L.L.) We thank the United States Geological Survey (USGS) and NOAA/GLERL for providing access to environmental metabolomics datasets used in this study.