Ion-networks are a complete yet sparse representation of an MS experiment containing multiple samples. It was originally developed for LC-IMS-MS DIA data, but the IMS dimension can easily be replaced by e.g. a scanning quadrupole coordinate, since precursor m/z and drift time are correlated. The original paper describing ion-networks is available on bioRxiv and all original data is available at PRIDE PXD015318.

The current implementation allows to

1. Create ion-networks
2. Annotate ion-networks (proteomics only)
3. Browse ion-networks

The complete software suite was developed for Linux. As Windows 10 nowadays comes with a Linux subsystem (WSL), this platform is compatible as well through a slightly modified installation. All source code was written in Python 3.6 and tested on Ubuntu 18.04 and CentOS 7.6.1810. The installation includes the following:

• The full software suite to Create/Annotate/Browse ion-networks.
• Some standard indexed proteomic databases (1GB).
• Percolator and Elude.
• A full demo proteomics benchmark dataset (3GB), containing an excerpt from PXD001240 with 10 minutes from 5 samples in condition A (65% human, 15% yeast, 20% ecoli), and 5 samples in condition B (65% human, 30% yeast, 5% ecoli). This test dataset allows to recreate the main figure of our paper, although some minor differences are present as the automatic estimation, automatic calibration and Percolator can only use the included data instead of the complete experiment.

Windows users need to install a WSL for Ubuntu 18.04 by following these steps. After the WSL has been installed and a user account has been created, copy-paste and execute the commands below line-per-line. Note that the second line takes about 30 minutes and several confirmations requiring you to manually type y. Once the auto-reboot screen prompts, you can safely click yes as this concerns only a WSL reboot without affecting Windows.

sudo -i
sudo apt update && sudo apt upgrade && sudo apt install python3.6 python3.6-venv python3.6-tk python3 python3-venv python3-tk libgomp1
mkdir percolator
wget --output-document percolator/percolator_3_02_01_ubuntu.tar.gz https://github.com/percolator/percolator/releases/download/rel-3-02-01/ubuntu64.tar.gz
tar xzvf percolator/percolator_3_02_01_ubuntu.tar.gz -C percolator
sudo dpkg -i percolator/elude-v3-02-linux-amd64.deb
sudo dpkg -i percolator/percolator-v3-02-linux-amd64.deb
rm -r percolator
wget https://github.com/swillems/histopya/archive/version_0.1.191028.tar.gz
tar xzvf version_0.1.191028.tar.gz
mv histopya-version_0.1.191028 histopya
cd histopya
bash install/install.sh
chmod -R 777 .

Next, close the WSL and download and install MobaXterm v11.1. Open it and press the session button on the top left. Select the rightmost tab WSL and set the Linux distribution to Ubuntu in the Basic WSL settings tab. Click the Advanced WSL settings tab and copy cd histopya; bash run_gui.sh to the Execute the following commands at startup window. Finally, click the Bookmark settings tab and change the Session name to e.g. ion_network_gui. Click the Create a desktop shortcut to this session button and select both options Hide terminal on startup and Close MobaXterm on exit before pressing OK in this popup. Confirm the session settings with OK. A pop-up with the GUI running should have appeared in your taskbar, allowing you to test the installation. To this end, click the select parameters button and subsequently browse in the Select parameter file pop-up. Navigate to the install folder and select the test_lfq_parameters.json file. Finally, perform a full test of all features with the buttons Create/Annotate/Browse ion-network. Each of them will show a pop-up that should complete with the final message Finished external script, indicating you can proceed with the next step. If the final Browse ion-network step correctly shows a second pop-up in which you can browse the test ion-network, you have successfully installed the complete software suite and can close MobaXTerm. For subsequent use, there is desktop-item that allows you to run the ion_network_gui with just a simple double-click.

NOTE: The WSL is a hidden subfolder located at e.g. C:\Users\sanwill\AppData\Local\Packages\CanonicalGroupLimited.Ubuntu18.04onWindows_79rhkp1fndgsc\LocalState\rootfs\home\histopya. Vice-versa, your windows drives are accessible from within your WSL through the location /mnt/. While you can copy-paste between your windows WSL and Windows 10, there might be some issues with permissions.

First, run the following commands to install all dependencies with e.g. apt and dpkg on Ubuntu 18.04 (skip this if already satisfied):

sudo apt update && sudo apt upgrade && sudo apt install python3.6 python3.6-venv python3.6-tk python3 python3-venv python3-tk libgomp1
mkdir percolator
wget --output-document percolator/percolator_3_02_01_ubuntu.tar.gz https://github.com/percolator/percolator/releases/download/rel-3-02-01/ubuntu64.tar.gz
tar xzvf percolator/percolator_3_02_01_ubuntu.tar.gz -C percolator
sudo dpkg -i percolator/elude-v3-02-linux-amd64.deb
sudo dpkg -i percolator/percolator-v3-02-linux-amd64.deb
rm -r percolator

Next, install the source for for ion-networks itself:

wget https://github.com/swillems/histopya/archive/version_0.1.191028.tar.gz
tar xzvf version_0.1.191028.tar.gz
mv histopya-version_0.1.191028 histopya
cd histopya
bash install/install.sh
chmod -R 777 .

Finally, test if it is correctly installed by Creating/Annotating/Browsing the demo dataset with:

bash run_cmd.sh -i install/test_lfq_parameters.json -a CAB

To create an ion-network, nothing more is needed than a single folder containing a csv per run of the experiment with all its peak picked fragment ions. Herein, each ion needs to have the following attributes:

1. m/z
2. DT
3. RT
4. intensity
5. m/z error (ppm)
6. DT error
7. RT error

In case of Waters data (e.g. HDMSE from a Synapt G2-Si or SONAR from a Vion), peak picked data is most easily obtainable by Waters' Apex3D software as provided by the commercial software packages MassLynx or Progenesis QI (Nonlinear Dynamics) with a command similar to:

Apex3D64.exe -pRawDirName sample.raw -outputDirName peak_picked_sample_folder -lockMassZ2 785.8426 -lockmassToleranceAMU 0.25 -bCSVOutput 1 -writeFuncCsvFiles 0 -leThresholdCounts 1 -heThresholdCounts 1 -apexTrackSNRThreshold 1 -bEnableCentroids 0

Alternatively, the freely available IMTBX and Grppr can be used for Waters' peak picking but this has not been tested for compatibility yet.

In case of proteomics data, a database is needed for fragment annotation. Human, Yeast, Ecoli and benchmark databases are included in the lib/databases/ folder. Custum databases can be created with some simple coding (src/peptides.py script), but no GUI has been provided yet.

Analysis of ion-networks can be done both by the command-line and with a graphical user interface (GUI). To use the command-line, a .json file needs to be created (either manual or with the GUI) which should at least contain the following parameters:

{
    "APEX_PATH": "data/test_lfq/",
    "OUTPUT_PATH": "projects/test_lfq/",
    "DATABASE_FILE_NAME": "lib/databases/lfq_benchmark.hdf5"
}

With this parameter file, three different actions can (simultaneously) be taken with the command-line flag -a or by simple selection in the GUI.

An ion-network can be created with the following command:

bash run_cmd.sh -i install/test_lfq_parameters.json -a C

While the default parameters generally suffice, the following parameters can have a significant effect:

• "NEIGHBOR_THRESHOLD": 0.997 Proxy for robustness. Higher values have less deconvoluting power, but higher robustness. Set closer to 1 for experiments with many samples.
• "SIGNAL_COUNT_THRESHOLD": 2 Minimum reproducibility for an aggregate, set higher for more stringent denoising.

Currently, only proteomics data can be annotated with a database search approach. Human, Yeast, Ecoli and benchmark databases are available, but more can be created by the user with some custom coding.

To annotate an ion-network, use the following command:

bash run_cmd.sh -i install/test_lfq_parameters.json -a A

Significant parameters that can change annotation results include:

• "USE_RT_IN_PERCOLATOR": false Use Elude to improve Percolator. Improves annotation results, but can take a lot of time.
• "REQUIRE_PHYSICAL_PRECURSOR": false If set to true, an unfragmented precursor signal is required to consistently co-elute before an annotation can be considered.
• "IDENTIFICATION_PPM": 20 Only fragments within this error range can be annotated.

An (un)annotated ion-network can be visualized with:

bash run_cmd.sh -i install/test_lfq_parameters.json -a B

Note that most ion-networks are quite vast and that it is not always wise to try and visualize everything. This is particularly true for the button Show edges and the slider Minimum signal, which generally should only be changed when zoomed in sufficiently.

Aggregates can be selected by double clicking and multiple aggregates can be selected by ctrl-double-click. Note that there is a minor bug where double clicking can force zooming when this option is still active, so we advise to first zoom to a region of interest, then turn off the zoom function and only then to start selecting aggregates of interest.