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prophosqua

Integration of phosphoproteome and total proteome data for comprehensive PTM analysis

The prophosqua package provides tools for integrating and analyzing post-translational modification (PTM) data with total proteome measurements. It enables researchers to distinguish between changes in protein abundance and changes in modification site usage, providing deeper insights into cellular signaling and regulation.

Overview

The integrated PTM analysis is carried out using prophosqua, which implements three complementary statistical approaches:

  • DPA (Differential PTM Abundance): tests for changes in PTM-site intensity between conditions.
  • DPU (Differential PTM Usage): tests whether the ratio of PTM-site to total protein intensity changes, identifying regulation independent of protein abundance.
  • CorrectFirst: applies protein-level correction before testing PTM sites, an alternative approach to distinguish PTM-specific regulation from protein expression changes.

Building on the prolfqua and prolfquapp packages, prophosqua additionally provides:

  • N-to-C plots - Visualization of phosphorylation sites along protein backbones
  • Sequence logo analysis - Identification of kinase recognition motifs
  • PTM-SEA - Post-translational modification set enrichment analysis
  • Kinase activity inference - Kinase library-based analysis from phosphoproteomics data
  • Motif enrichment analysis - MEA visualization
  • Enrichment visualization - Dot plots, heatmaps, and volcano plots for enrichment results

Installation

Prerequisites

This package depends on several other packages that should be installed first:

# Install prolfqua (core proteomics analysis package)
library(devtools)
devtools::install_github('protviz/prozor', dependencies = TRUE)
devtools::install_github('prolfqua/prolfqua', dependencies = TRUE)
# Install prolfquapp (proteomics analysis workflow package)
devtools::install_github('prolfqua/prolfquapp', dependencies = TRUE, build_vignettes=TRUE)

For detailed installation instructions and system requirements, see: - prolfqua GitHub repository - prolfquapp GitHub repository

Install prophosqua 

library(devtools)
devtools::install_github('prolfqua/prophosqua', dependencies = TRUE, build_vignettes=TRUE)

Usage

Basic Workflow

  1. Run differential expression analysis using prolfquapp for both total proteome and PTM data
  2. Integrate the results using prophosqua::test_diff()
  3. Visualize the data with N-to-C plots and sequence logos
  4. Generate comprehensive reports with statistical summaries

Example 

library(prophosqua)

# Load your data
tot_res <- load_and_preprocess_data(total_proteome_file, required_cols)
phospho_res <- load_and_preprocess_data(ptm_file, required_cols)

# Integrate analysis
combined_test_diff <- test_diff(phospho_res, tot_res, join_column = join_column)

# Create visualizations
plot_data <- n_to_c_usage(combined_test_diff, contrast_name, FDR_threshold = 0.05)

Vignettes

The package includes vignettes demonstrating the analysis workflow:

  • MiMBIntegratedPTM.Rmd - Integrated analysis of PTM and total proteome (DPA, DPU, CorrectFirst)
  • Analysis_n_to_c.Rmd - N-to-C plots for PTM site visualization
  • Analysis_seqlogo.Rmd - Sequence logo analysis
  • Analysis_PTMSEA.Rmd - PTM-SEA analysis
  • Analysis_KinaseLibrary.Rmd - Kinase activity inference from phosphoproteomics data
  • Analysis_MEA.Rmd - Motif enrichment analysis visualization
  • QCReport.qmd - FragPipe TMT quality control report

Citation

If you use this package in your research, please cite:

Wolski W, Dittmann A, Panse C, Kunz L, Grossmann J. “Integrated Analysis of Post-Translational Modifications and Total Proteome: Methods for Distinguishing Abundance from Usage Changes.” Methods in Molecular Biology, 2025 (submitted).

Grossmann J, Wolski W. “prolfqua/prophosqua: 0.1.0.” Zenodo, 2025. DOI: 10.5281/zenodo.15845272

  • prolfqua - Core proteomics analysis package
  • prolfquapp - Proteomics analysis workflow package

Building and Deploying Documentation

Build pkgdown site locally 

pkgdown::build_site()

Deploy to GitHub Pages 

# Using ghp-import (via uv)
uvx ghp-import -n -p -f docs

Then enable GitHub Pages in repo settings (Settings -> Pages -> Source: gh-pages branch).

Site: https://prolfqua.github.io/prophosqua

Contributing

Contributions are welcome! Please visit our GitHub repository for: - Issue reporting - Feature requests - Code contributions

License

This package is released under the MIT License.