proteoCraft

A set of scripts and functions for R-based MS-Proteomics analysis.

The following types of experiments are covered:

1. Replicates available (2 workflows: protein groups or modified peptides only):
   • "Regulation analysis", aka DEP (Differential Expression of Proteins)
   • Pull down analysis (co-IPs, BioID, TurboID, APEX...), in which case also saintEXPRESS analysis is performed
   • Regulation analysis of post-translationally modified peptides
   • Analysis of changes to subcellular localisation
   • Time course analysis
2. No replicates available (1 workflow):
   • Samples characterisation
   • Gel band ID
   • Basic, no-statistics involved comparisons between samples

(NB: All workflows could previously be run through a single master script, which ran code chunks sequentially and allowed partial processing. It is still included, but hasn't been tested/maintained in a while, so we would recommend not to use it for now.)

Input data

Though where possible the scripts have been written as generally as possible, they have only been tested on data from:

• ThermoScientific Q-Exactive HF (a few datasets on Exploris with FAIMS or Fusion Tribrid)
• Bruker timsTOF HT Input data can be the output from:
• MaxQuant
• diaNN
• FragPipe (using "reasonable" search parameters... FragPipe is very flexible) There is burgeoning support for:
• Proteome Discoverer (conversion function exists but integration with analysis scripts deprecated)
• Skyline (new basic conversion function in the works, probably needs fuller development and integration with the scripts but for now allows to deal with .tsv/.csv exports assuming a core of columns were exported)

Depending on search parameters you may encounter unexpected situations, as we have not tested every possible situation but only ones occurring in our facility. It is likely that some options in some software will break the conversion functions, especially for the more versatile engines such as FragPipe. If that happens, get in touch and we will add support asap. We would like to add Spectronaut/Peaks/AlphaPept support too eventually.

Environment and requisites

This is meant to run through RStudio in Windows and will probably not work without significant edits on Linux/Unix. This is because the scripts will sometimes:

• use R code which is Windows-specific, e.g. makes assumptions about the type of UI/GUI available.
• build and run windows command line or powershell-specific commands. Run this ideally on a multi-core PC optimized for data analysis with a lot of memory. Many steps are parallelized. We run tests on a Windows Server PC with 56 vCPUs and 128 GB RAM.

In addition to R and RStudio, for a smoother experience you should also install:

• ScanHeadsMan (run by MatMet_LCMS, extracts methods from Thermo raw files)
• Cytoscape (for ClueGO GO-terms enrichment analysis and network visualisation)
• Word or any software which can open .docx files (to check the Materials and Methods template text)
• Excel or any software which can open .xlsx files (to check the final Report file)
• Optional: a web browser for opening interactive plots (.html files) Normally, the scripts provided should automatically install any R packages required. In case this fails just manually install the missing ones.

Troubleshooting:

• In our hands RStudio can become very slow after some time. To solve the issue, go to Tools > Global Options..., click on Code then Diagnostics and untick "Show diagnostics for R"
• Up to v_6.4.0.0, some shiny apps in these workflows (specifically filling down dropdown menu selections) did not work if shiny_1.8.0 or later was used. This should be fixed now, and with the way the code was rewritten should also be faster and still work with older version. Still, kt keep an eye on it...

Note on speed:

This package has been somewhat optimised for speed, but many steps could still probably be faster. The current main bottlenecks include: - Extracting MS methods using ScanHeadsMan, - Checking peptide-to-protein assignments provided by the search software (recommended), - Extracting TIC/Base peak information from Thermo files using the rawrr package, - Cytoscape-dependent steps.