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Prototype of PNNL pipeline

Testing implementation of PNNL pipeline on BIC infrastructure

Schema

Initial documents provided by the PNNL with details of the pipeline (converted to md format by the BIC) :

The scripts directory contains additional files and scripts.

WORKFLOW

GLOBAL PROTEOME Test files

Two raw files used for testing purposes from the pilot project global protein abundance dataset:

MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02.raw
MoTrPAC_Pilot_TMT_W_S1_02_12Oct17_Elm_AQ-17-09-02.raw

PHOSPHO PROTEOME Test files

Two raw files used for testing purposes from the pilot project phospho protein abundance dataset:

MoTrPAC_Pilot_TMT_P_S1_01_DIL_28Oct17_Elm_AQ-17-10-03.raw
MoTrPAC_Pilot_TMT_P_S2_01_3Nov17_Elm_AQ-17-10-03.raw

OVERVIEW

Step Description Software Input Output Shell Script (Global) Shell Script (Phospho)
00 Extract reporter ion peaks from MS2 spectra and create Selected Ion Chromatograms for each MS/MS parent ion MASIC Thermo .Raw file _ReporterIons.txt and _SICStats.txt (plus several other files) step00masic.sh step00masic_phospho.sh
01 Convert Thermo .raw files to .mzML files (standard XML file format for MS data) MSConvert Thermo .Raw file .mzML file step01convertRaw.sh step01convertRaw_phospho.sh
02 Identify peptides using a fully tryptic search (for speed); these are used in Step 03 MS-GF+ .mzML file and .fasta file .mzid file step02msgfplus_tryptic.sh step02msgfplus_tryptic_phospho.sh
03a Use mass error histograms to in-silico re-calibrate the m/z values in the .mzML file mzrefiner filter in MSConvert .mzid file and .mzML file _FIXED.mzML step03a.sh step03a_phospho.sh
03b Plot the mass error histograms before and after in-silico recalibration PPMErrorCharter .mzid file and _FIXED.mzML file PNG files step03b.sh step03b_phospho.sh
04 Identify peptides using a partially tryptic search MS-GF+ _FIXED.mzML file and .fasta file .mzid file step04msgfplus.sh step04msgfplus_phospho.sh
05 Create a tab-separated value file listing peptide IDs from step 04 MzidToTSVConverter .mzid file .tsv file step05net462.sh step05net462_phospho.sh
06 Create tab-delimited files required for step 7; files contain peptide IDs, unique sequence info, and residue modification details PeptideHitResultsProcessor .tsv file _syn.txt file and several related files step06phrp.sh step06phrp_phospho.sh
07 Localize the position of Phosphorylation on S, T, and Y residues in phosphopeptides Ascore _syn.txt files, _FIXED.mzML file, .fasta file _syn_plus_ascore.txt file n/a step07ascore_phospho.sh

DETAILS

Notes on containers

Check this file for details about containers (build, tag, and push to container registry). All the steps described below are based on "interactive" mode. To directly run the scripts in the containers follow the steps on this file.

Step 00: MASIC

Build Docker image (one time, or after a new version of MASIC is released):

Start the container (interactive mode)

docker run -v $PWD/data:/data:rw -v $PWD/parameters:/parameters:rw -v $PWD/step00:/step00:rw -it motrpac-prot-masic:v1.2_20200901 /bin/bash

Run in docker:
step00masic.sh
step00masic_phospho.sh

docker exec motrpac-prot-masic step00/step00masic.sh

Step 01: Convert .raw to .mzML files

step01convertRaw.sh
step01convertRaw_phospho.sh

  • Input directory/files: /data/test_global/raw/*.raw
  • Output directory: msgfplus_input/*.mzML

Step 02: Identify peptides using a fully tryptic search

Run on Docker MS-GF+ using the .mzML file from msconvert (step 1), get a .mzid file

Run MS-GF+ on Docker container (openjdk). Created a Dockerfile available in directory step02.

Start the container (interactive mode)

docker run -v $PWD/data:/data:rw -v $PWD/parameters:/parameters:rw -v $PWD/step02:/step02:rw -it motrpac-prot-msgfplus:v1.2_20200901  /bin/bash

Run in docker:
step02msgfplus_tryptic.sh
step02msgfplus_tryptic_phospho.sh

  • Input directory/files: /data/test_global/msgfplus_input/
    • *.mzML
    • sequence_db.fasta sequence db
    • MSGFPlus_Mods.txt config file
  • Output directory/file: /data/test_global/msgfplus_output/*.mzid

Step 03: Use mass error histograms to in-silico re-calibrate the m/z values

A) Run msconvert with the mzrefiner option to create a new .mzML file named _FIXED.mzML

step03a.sh
step03a_phospho.sh

ISSUE: msconvert ignores the path to the specified output directory

B) Run PPMErrorCharter using the .mzid file from step 2a and the _FIXED.mzML file from step 3a

Start the container:

docker run -v $PWD/data:/data:rw -v $PWD/parameters:/parameters:rw -v $PWD/step03:/step03:rw -it prot-ppmerror:v1.2.7632c /bin/bash

Run in docker:
step03b.sh
step03b_phospho.sh

Step 4: Protein Identification and Quantification

Run MS-GF+ using the _FIXED.mzml file from Step 3: That creates a .mzID file (called it Dataset_final.mzid).

Run the same docker container as in step02

docker run -v $PWD/data:/data:rw -v $PWD/parameters:/parameters:rw -v $PWD/step04:/step04:rw -it motrpac-prot-msgfplus:v1.2_20200901  /bin/bash

Run in docker:
step04msgfplus.sh
step04msgfplus_phospho.sh

Step 5: MzidToTSVConverter

Run MzidToTSVConverter to convert Dataset_final.mzid to Dataset.tsv

Start the container:

docker run -v $PWD/data:/data:rw -v $PWD/parameters:/parameters:rw -v $PWD/step05:/step05:rw -it motrpac-prot-mzid2tsv:v1.2_20200901 /bin/bash

Run in docker:
step05net462.sh
step05net462_phospho.sh

Step 6: PeptideHitResultsProcRunner

Run PeptideHitResultsProcRunner using the .tsv file from step 5:

Start the container:

docker run -v $PWD/data:/data:rw -v $PWD/parameters:/parameters:rw -v $PWD/step06:/step06:rw -it motrpac-prot-phrp:v1.2_20200901 /bin/bash

Run in docker:
sh step06phrp.sh
sh step06phrp_phospho.sh

Step 7: AScore

Start the container:

docker run -v $PWD/data:/data:rw -v $PWD/parameters:/parameters:rw -v $PWD/step07:/step07:rw -it motrpac-prot-ascore:v1.2_20200901 /bin/bash

Run in docker:
step07ascore_phospho.sh

Relative Quantification: PlexedPiper

PlexedPiper repo

Start container:

docker run -v $PWD/data:/data:rw -v $PWD/relquant:/relquant:rw  -it motrpac-prot-plexedpiper:v1.2_20200901 /bin/bash
docker run -v $PWD/relquant:/relquant:rw  prot-plexedpiper:v0.1.20201104 /bin/bash
docker run -it gcr.io/motrpac-portal/prot-plexedpiper:v0.1.20201104 /bin/bash

Run in docker: \

sh relquant.sh
sh relquant_phospho.sh

Notes

MSConvert.exe: Linux version is available here. Example command line they have using docker:

docker run -it --rm -e WINEDEBUG=-all -v /your/data:/data chambm/pwiz-skyline-i-agree-to-the-vendor-licenses wine msconvert /data/file.raw