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Phospho-Analyst
A tool for analysing label-free quantitative proteomics dataset on both peptide and protein level https://analyst-suites.org/apps/phospho-analyst/
![TMT-analyst_pipeline](https://github.com/MonashProteomics/Phospho-Analyst/raw/main/www/Phospho_Analyst.svg)
Motivation
- Automate downstream statistical analysis of Label free quantitative proteomics data on both peptide and protein level (generated by MaxQuant)
Input
- MaxQuant Phospho (STY)Sites.txt file
- A phosphosite experiment design table (tab separated file) containing three columns ("label", "condition", "replicate")
- (Optional) MaxQuant proteinGroups.txt file
- (Optional) A protein experiment design table (tab separated file) containing three columns ("label", "condition", "replicate")
Data pre-filtering criteria
-Remove reverse sequences
-Remove potential contaminants
-Remove localization probability < 0.75
-Expand Phosphosite intensity columns from wide to long format
-Extract peptide sequences information
-Remove observations with high proportion of missing values
Advanced parameters to choose
- Differencial expression cutoff
- Adjusted p-value cutoff (FDR cutoff on quantitation)
- Log2 fold change cutoff
- Option to choose paired test for matched pair data
- Types of imputation
- A number of missing value imputation options including knn, Minpob etc.
- Type of FDR correction
- Benjamin Hochberg (BH) method
- t-statistics correction: Implemented in fdrtools
- Option to include phosphosites/proteins identified/quantified with a single unique peptide.
- Select how many clusters of differentially expressed phosphosites/proteins needed for the heatmap (default is 6)
Outputs
Result table
- Results Table: Includes names (Phosphosite/Gene names), Phosphosite/Protein Ids,
Adjusted ANOVA p-values, ANOVA p-values,Log
fold changes/ ratios (each pairwise comparisons), Adjusted
p-values (applying FDR corrections), p-values, Boolean values
for significance, average protein intensity (log transformed) in
each sample.
Result Plots
- Interactive volcano plot for each pairwise comparison.
- Heatmap of differencially expressed phosphosites/proteins
- Phosphosite intensity plots for a single or group of selected phosphosites/proteins from table.
QC Plots
- PCA plot (Could move to QC section)
- Sample Correlation (pearson correlation)
- Sample Coefficient of variations (CVs)
- Number of phosphosites/proteins per sample
- Sample coverage (overlap of identified phosphosites/proteins across every sample)
- Missing value heatmap
- Imputation effect on sample distribution
Download options
Download tables (csv format)
- Results: Same as Results Table
- Unimputed data matrix: Original phosphosite/protein intensities before
imputation in each sample.
- Imputed data matrix: Phosphosite/Protein intensities after performing selected
imputation method
- Full results: Combined table of all above data outputs i.e. with and
without imputation information, along with fold change and p-values.
Download Report
- A summary report for each analysis that
includes method, summary statistics and plots.
Local installation
The current version of Phospho-Analyst is hosted on R - 4.2.1
. The required packages can be installed by running the install.R.
Once installed all the dependencies following steps to run the server locally.
Move to the folder
cd Phospho-Analyst
Inside R console or R studio
library("shiny")
runApp()
- Using Docker
Install & start Docker demon on your PC
Option one:
Pull Phospho-Analyst image from Docker Hub (From terminal)
docker pull haileyzhang/phospho-analyst:tagname
Option two:
Clone the repository
git clone https://github.com/MonashProteomics/Phospho-Analyst.git
Move to the folder
cd Phospho-Analyst
Build Phospho-Analyst (Any name after -t)
docker build -f Dockerfile -t phospho-analyst .
Run Phospho-Analyst (From terminal)
docker run -p 3838:3838 phospho-analyst
Open local interface
https://localhost:3838/phospho-analyst