Complete Computational Workflow for Integrated PTM and Total Proteome Analysis: Distinguishing Expression from Usage Changes

Complete Computational Workflow for Integrated PTM and Total Proteome Analysis: Distinguishing Expression from Usage Changes # PTM Analysis Example - Maculins et al. 2020 Example dataset and analysis outputs for integrated phosphoproteomics analysis using the `prophosqua` R package. ## Main Document **[Supplementary_Material_v3.html](Supplementary_Material_v3.html)** - Complete walkthrough of the integrated phosphoproteomics analysis pipeline, including: - Data preprocessing and quality control - Differential expression analysis (total proteome and phospho) - Differential PTM Abundance (DPA) and Differential PTM Usage (DPU) analysis - Sequence motif analysis - Result visualization and interpretation The source file `Supplementary_Material_v3.Rmd` can be adapted for your own datasets. ## Dataset **Atg16l1 macrophage study** (Maculins et al., eLife 2020) investigating the role of autophagy in bacterial infection responses. - **Experiment**: TMT-11-plex quantitative proteomics - **Design**: 2x3 factorial - Genotype: Wild-type (WT) vs Atg16l1-knockout (KO) - Timepoint: Uninfected, Early infection, Late infection - **Biological context**: Macrophage responses to *Shigella* infection - **Data types**: Total proteome + phospho-enriched samples ## Directory Structure ``` PTM_example_analysis_v2/ ├── PTM_example/ # Raw FragPipe outputs │ ├── data_total/FP_22/ # Total proteome PSM files │ └── data_ptm/FP_22/ # Phospho-enriched PSM files ├── DEA_*_vsn/ # Differential expression analysis │ ├── Results_WU_*/ # Analysis results (Excel, HTML reports) │ └── Inputs_WU_*/ # Input files and Rmd templates ├── PTM_analysis_*/ # Integrated PTM/protein analysis │ ├── Result_phosphoAndTotalIntegration.xlsx │ ├── Result_phosphoAndTotalIntegration.html │ ├── Site_differential_Expression_multicontrast.pdf │ └── Site_differential_UsageChange_multicontrast.pdf ├── Supplementary_Material_v3.html # Main analysis document ├── Supplementary_Material_v3.Rmd # Source (adapt for your data) ├── QCReport.html # TMT labeling efficiency QC report ├── QCReport.qmd # QC report source ├── _Overview_PhosphoAndIntegration_site.html # Interactive integration report ├── dataset_with_contrasts.tsv # Sample annotation with contrasts └── config.yaml # Analysis configuration ``` ## Key Output Files | File | Description | |------|-------------| | `Supplementary_Material_v3.html` | **Main document** - Complete analysis walkthrough | | `QCReport.html` | TMT labeling efficiency, missed cleavages, channel balance | | `_Overview_PhosphoAndIntegration_site.html` | Interactive PTM/protein integration report | | `DEA_*/Results_*/DE_*.xlsx` | Differential expression statistics | | `PTM_analysis_*/Result_phosphoAndTotalIntegration.xlsx` | Integrated DPA/DPU results | ## Contrasts Analyzed 1. **KO_vs_WT** - Main effect across all timepoints 2. **KO_vs_WT_at_Uninfect** - Genotype effect in uninfected condition 3. **KO_vs_WT_at_Early** - Genotype effect at early infection 4. **KO_vs_WT_at_Late** - Genotype effect at late infection ## Citation Maculins T, et al. (2020). Bacteria-host relationship: ubiquitin ligases as weapons of invasion. *eLife* 9:e62320. DOI: [10.7554/eLife.62320](https://doi.org/10.7554/eLife.62320) ## Rendering QCReport The `QCReport.qmd` vignette generates TMT labeling QC reports. To render it: ```r # From vignettes/ directory cd vignettes/ Rscript -e "rmarkdown::render('QCReport.qmd')" # Or using snakemake snakemake -j1 render_qc ``` The report automatically downloads example data (Arabidopsis qc_example_data) from GitLab. For custom data, provide parameters: ```r rmarkdown::render('QCReport.qmd', params = list( wd = "path/to/working/dir", psm = "relative/path/to/psm.tsv", fasta = "relative/path/to/database.fasta", workunit = "your_workunit_id", projectid = "your_project_id" )) ``` ## Related Resources - [prophosqua R package](https://github.com/prolfqua/prophosqua) - [prolfquapp R package](https://github.com/prolfqua/prolfquapp) - [Zenodo Archive: Complete Computational Workflow for Integrated PTM and Total Proteome Analysis](https://doi.org/10.5281/zenodo.15830988)