DOI: 10.5281/zenodo.15830988
title: Complete Computational Workflow for Integrated PTM and Total Proteome Analysis: Distinguishing Expression from Usage Changes

description: 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)