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DIY: Using MS²PIP predicted spectral libraries for DIA data extraction

In close collaboration with the CompOmics group of Lennart Martens we worked out a complete workflow to use MS²PIP predicted libraries for wide window DIA data extraction.

This document outlines all required steps to try out the analysis yourself. In the methods section of our preprint The future of peptide-centric Data-Independent Acquisition is predicted, you can find a more detailed description of all steps.

Installation

For this manuscript, a Linux operated machine with a Python 3 virtual environment was used. If you do not have access to a server or Linux operated machine, we recommend you to install the VirtualBox software to create a virtual clone on your Windows/macOS operated PC. We can also highly recommend the Windows Subsystem for Linux which is implemented on all Windows 10 PCs.

MS²PIP and fasta2speclib

MS²PIP can be downloaded from GitHub page of MS²PIP. A detailed installation guide can be found on Extended Install Instructions.

Elude

Elude is shipped with Percolator. It can be downloaded from the Percolator GitHub repository. To install Elude, after unzipping the folder, run the following command:

sudo dpkg -i elude-v3-02-linux-amd64.deb (Linux operated machine) | dpkg -i  Ubuntu64.deb

EncyclopeDIA

EncyclopeDIA can be downloaded from https://bitbucket.org/searleb/encyclopedia/downloads/?tab=downloads.

Run the pipeline

Input files

  • The Yeast dataset itself can be downloaded from the MassIVE proteomics repository: MSV000082805. The repository contains both raw as peak picked data, so it is up to you whether you want to skip the raw file processing (e.g. with MSConvertGUI), or not.
  • We shared the yeast FASTA file on this repository: DIY/YEAS8_iRT.fasta. It can also be downloaded from UniProt.
  • The Elude model we used can also be found on this repository: ELUDE/ELUDE_PAN_HUMAN.model
  • The fasta2speclib config file we used can be found here: Config_file/fasta2speclib_config_HCD_2missed_ELUDE.json

fasta2speclib

Once everything is installed you can start predicting spectral libraries using one single command:

python3 “fasta2speclib.py” [-h] [-o OUTPUT_FILENAME] [-c CONFIG_FILENAME] “fasta_filename”

More documentation on fasta2speclib can be found on the MS²PIP wiki pages.

If you want to skip this step, you can download the predicted spectral libraries (Human, Yeast) in .dlib format on genesis.ugent.be/uvpublicdata/MS2PIP-for-DIA/

EncyclopeDIA

Using the EncyclopeDIA CONVERT MSP/SPTXT to Library function, you can convert the fasta2speclib .msp files to a .dlib file. This .dlib file can then be used in the EncyclopeDIA analysis to make a chromatogram library. Next, the wide window runs can be analyzed with the chromatogram library.

Contact

If you would have any issues, please contact us and we will try to help you wherever we can.