Phospho-Analyst

A tool for analysing label-free quantitative proteomics dataset on both peptide and protein level https://analyst-suites.org/apps/phospho-analyst/

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

1. Interactive volcano plot for each pairwise comparison.
2. Heatmap of differencially expressed phosphosites/proteins
3. Phosphosite intensity plots for a single or group of selected phosphosites/proteins from table.

QC Plots

1. PCA plot (Could move to QC section)
2. Sample Correlation (pearson correlation)
3. Sample Coefficient of variations (CVs)
4. Number of phosphosites/proteins per sample
5. Sample coverage (overlap of identified phosphosites/proteins across every sample)
6. Missing value heatmap
7. Imputation effect on sample distribution

Download options

Download tables (csv format)

1. Results: Same as Results Table
2. Unimputed data matrix: Original phosphosite/protein intensities before imputation in each sample.
3. Imputed data matrix: Phosphosite/Protein intensities after performing selected imputation method
4. 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.

• Using git and Rstudio

  
  ## Clone the repository
  git clone https://github.com/MonashProteomics/MaxQuant-phosphoproteomics-analysis.git

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