Since others are going to start doing proteomics analysis, I figured I would take a bit of time to update the workflow, mostly to remove all of the installation and troubleshooting and to just show how to different programs work and interact.
As with other things I’ve done, this is all done in R, if you’d like to work through either command line, or Jupyter notebook, you can replace the system() calls with print(), which will cause R to just output the terminal commands to the console.
Step 1: Convert .raw to .mzXML.
setwd("/home/shared/ReAdW")
system("WINEPREFIX=~/.wine32 wine ReAdW.2016010.msfilereader.exe ~/Documents/ProteomicsTest/20161205_sample_1.raw ~/Documents/ProteomicsTest/20161205_sample_1.mzXML")fixme:thread:GetThreadPreferredUILanguages 52, 0x32fafc, 0x32fc74 0x32fb04
fixme:heap:HeapSetInformation (nil) 1 (nil) 0ReAdW 2016.1.0(build Jun 21 2016 11:13:06)
Xcalibur interface initialized (5).
(Thermo lib opened file /home/srlab/Documents/ProteomicsTest/20161205_sample_1.raw)
file version is 66, interface version is 2.0 or greater
file should contain scan numbers 1 through 109165
(got computer name: emu)
Calculating sha1-sum of mzXML
--done (mzXML sha1):ce63ef716956b4ba6640cafdc77c1dc027153326Huzzah! We now have a file that works in Comet.
Step 2: Comet
I used a Gigas Proteome from Uniprot found here and a stock Comet params file from here
These files need to be in the same directory as your data files. The lack of spaces on the -P and -D arguments are also intentional.
setwd("~/Documents/ProteomicsTest")
system("/home/shared/comet/comet.2016012.linux.exe -Pcomet.params.high-low -Dgigas-proteome.fasta 20161205_sample_1.mzXML") Comet version "2016.01 rev. 2"
Search start: 12/27/2016, 10:50:13 AM
- Input file: 20161205_sample_1.mzXML
- Load spectra: 89352
- Search progress: 0% 1% 2% 3% 5% 6% 7% 7% 8% 9% 10% 10% 11% 12% 13% 13% 14% 15% 16% 16% 17% 18% 19% 20% 20% 21% 22% 22% 23% 24% 24% 25% 26% 27% 27% 28% 29% 29% 30% 31% 31% 32% 33% 33% 34% 35% 35% 36% 37% 38% 39% 39% 40% 41% 41% 42% 43% 43% 44% 45% 46% 46% 47% 48% 48% 49% 50% 51% 51% 52% 53% 54% 54% 55% 56% 57% 57% 58% 59% 59% 60% 61% 62% 62% 63% 63% 64% 65% 66% 67% 67% 68% 69% 70% 70% 71% 72% 72% 73% 74% 75% 75% 76% 77% 77% 78% 79% 80% 80% 81% 82% 82% 83% 84% 84% 85% 86% 87% 88% 89% 90% 91% 92% 93% 94% 95% 96% 97% 98% 99%
- Post analysis: done
Search end: 12/27/2016, 10:52:14 AMStep 3: xinteract from the Trans-Proteomic Pipeline (TPP)
Arguments are from Emma’s github post but Protein Prophet doesn’t find any decoys with the tag “DECOY_”, that may or may not be dataset/run specific?
system("/usr/tpp_install/tpp/bin/xinteract -dDECOY_ -N20161205_sample_1 20161205_sample_1.pep.xml -p0.9 -OAp")
/usr/tpp_install/tpp/bin/xinteract (TPP v5.0.0 Typhoon, Build 201612091438-exported (Linux-x86_64))
naming output file interact-20161205_sample_1.pep.xml
running: "/usr/tpp_install/tpp/bin/InteractParser 'interact-20161205_sample_1.pep.xml' '20161205_sample_1.pep.xml' -L'7'"
file 1: 20161205_sample_1.pep.xmlSUCCESS: CORRECTED data file /home/srlab/Documents/ProteomicsTest/20161205_sample_1.mzXML in msms_run_summary tag ...
SUCCESS: CORRECTED data file /home/srlab/Documents/ProteomicsTest/20161205_sample_1.mzXML in msms_run_summary tag ... processed altogether 89352 resultsINFO: Results written to file: /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xmlcommand completed in 26 sec
running: "/usr/tpp_install/tpp/bin/DatabaseParser 'interact-20161205_sample_1.pep.xml'"
command completed in 0 sec
running: "/usr/tpp_install/tpp/bin/RefreshParser 'interact-20161205_sample_1.pep.xml' 'gigas-proteome.fasta'" - Building Commentz-Walter keyword tree...
- Searching the tree...
- Linking duplicate entries...
- Printing results...
command completed in 10 sec
running: "/usr/tpp_install/tpp/bin/PeptideProphetParser 'interact-20161205_sample_1.pep.xml' DECOY=DECOY_ MINPROB=0.9 ACCMASS"
using Accurate Mass Bins
Using Decoy Label "DECOY_".
(Comet)
adding ACCMASS mixture distribution
init with Comet trypsin
MS Instrument info: Manufacturer: Thermo, Model: Orbitrap Fusion Lumos, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN
INFO: Processing standard MixtureModel ...
Initialising statistical models ... PeptideProphet (TPP v5.0.0 Typhoon, Build 201612091438-exported (Linux-x86_64)) AKeller@ISB
read in 0 1+, 38681 2+, 36514 3+, 9540 4+, 1967 5+, 0 6+, and 0 7+ spectra.
Found 0 Decoys, and 86702 Non-Decoys
WARNING: No decoys with label DECOY_ were found in this dataset. reverting to fully unsupervised method.Iterations: .........10.........20.........30...
model complete after 34 iterations
command completed in 68 sec
running: "/usr/tpp_install/tpp/bin/ProphetModels.pl -i interact-20161205_sample_1.pep.xml -d "DECOY_""
Analyzing interact-20161205_sample_1.pep.xml ...
Reading Accurate Mass Model model +1 ...
Reading Accurate Mass Model model +2 ...
Reading Accurate Mass Model model +3 ...
Reading Accurate Mass Model model +4 ...
Reading Accurate Mass Model model +5 ...
Reading Accurate Mass Model model +6 ...
Reading Accurate Mass Model model +7 ...
Parsing search results "/home/srlab/Documents/ProteomicsTest/20161205_sample_1 (Comet)"...
=> Found 34199 hits. (0 decoys, 0 excluded)
=> Total so far: 34199 hits. (0 decoys, 0 excluded)
command completed in 3 sec
running: "/usr/tpp_install/tpp/cgi-bin/PepXMLViewer.cgi -I /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml"
command completed in 3 sec
running: "/usr/tpp_install/tpp/bin/ProteinProphet 'interact-20161205_sample_1.pep.xml' 'interact-20161205_sample_1.prot.xml'"
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v5.0.0 Typhoon, Build 201612091438-exported (Linux-x86_64))
(no FPKM) (using degen pep info)
Reading in /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml...
...read in 0 1+, 17717 2+, 13325 3+, 2859 4+, 298 5+, 0 6+, 0 7+ spectra with min prob 0.05
Initializing 26114 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating protein lengths and molecular weights from database gigas-proteome.fasta
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000
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.........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000
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.........:.........:.........:.........:.........:.........:.........:.........:.........:........ Total: 25982
Computing degenerate peptides for 5078 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing 4730 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating sensitivity...and error tables...
Computing MU for 5262 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%INFO: mu=8.44703e-06, db_size=23302460
Finished. Results written to: /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.prot.xml
command completed in 19 sec
running: "/usr/tpp_install/tpp/bin/ProtProphModels.pl -i interact-20161205_sample_1.prot.xml"
Analyzing interact-20161205_sample_1.prot.xml ...
command completed in 0 sec
running: "/usr/tpp_install/tpp/bin/tpp_models.pl '/home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml'"
File: /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml
- in ms run: /home/srlab/Documents/ProteomicsTest/20161205_sample_1...
-------------------------------------------------------------------------------
TPP DASHBOARD -- started at Tue Dec 27 12:11:57 2016
-------------------------------------------------------------------------------
File /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml is pepxml
Found fval (+1) model...
Found ntt (+1) model...
Found nmc (+1) model...
Found AccurateMassModel ('+1') model...
Found IsoMassDiff (+1) model...
Found fval (+2) model...
Found ntt (+2) model...
Found nmc (+2) model...
Found AccurateMassModel ('+2') model...
Found IsoMassDiff (+2) model...
Found fval (+3) model...
Found ntt (+3) model...
Found nmc (+3) model...
Found AccurateMassModel ('+3') model...
Found IsoMassDiff (+3) model...
Found fval (+4) model...
Found ntt (+4) model...
Found nmc (+4) model...
Found AccurateMassModel ('+4') model...
Found IsoMassDiff (+4) model...
Found fval (+5) model...
Found ntt (+5) model...
Found nmc (+5) model...
Found AccurateMassModel ('+5') model...
Found IsoMassDiff (+5) model...
Found fval (+6) model...
Found ntt (+6) model...
Found nmc (+6) model...
Found AccurateMassModel ('+6') model...
Found IsoMassDiff (+6) model...
Found fval (+7) model...
Found ntt (+7) model...
Found nmc (+7) model...
Found AccurateMassModel ('+7') model...
Found IsoMassDiff (+7) model...
--> Trying to write file /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep-MODELS.html
-------------------------------------------------------------------------------
Finished at Tue Dec 27 12:12:01 2016 with 0 errors.
-------------------------------------------------------------------------------
command completed in 4 sec
running: "/usr/tpp_install/tpp/bin/tpp_models.pl '/home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.prot.xml'"
File: /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.prot.xml
-------------------------------------------------------------------------------
TPP DASHBOARD -- started at Tue Dec 27 12:12:01 2016
-------------------------------------------------------------------------------
File /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.prot.xml is protxml
Found end of header
--> Trying to write file /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.prot-MODELS.html
-------------------------------------------------------------------------------
Finished at Tue Dec 27 12:12:01 2016 with 0 errors.
-------------------------------------------------------------------------------
command completed in 0 sec
/usr/tpp_install/tpp/bin/InteractParser 'interact-20161205_sample_1.pep.xml' '20161205_sample_1.pep.xml' -L'7' 26 sec
/usr/tpp_install/tpp/bin/DatabaseParser 'interact-20161205_sample_1.pep.xml'
/usr/tpp_install/tpp/bin/RefreshParser 'interact-20161205_sample_1.pep.xml' 'gigas-proteome.fasta' 10 sec
/usr/tpp_install/tpp/bin/PeptideProphetParser 'interact-20161205_sample_1.pep.xml' DECOY=DECOY_ MINPROB=0.9 ACCMASS 68 sec
/usr/tpp_install/tpp/bin/ProphetModels.pl -i interact-20161205_sample_1.pep.xml -d "DECOY_" 3 sec
/usr/tpp_install/tpp/cgi-bin/PepXMLViewer.cgi -I /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml 3 sec
/usr/tpp_install/tpp/bin/ProteinProphet 'interact-20161205_sample_1.pep.xml' 'interact-20161205_sample_1.prot.xml' 19 sec
/usr/tpp_install/tpp/bin/ProtProphModels.pl -i interact-20161205_sample_1.prot.xml 0 sec
/usr/tpp_install/tpp/bin/tpp_models.pl '/home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml' 4 sec
/usr/tpp_install/tpp/bin/tpp_models.pl '/home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.prot.xml' 0 sec
job completed in 133 sec Step 4: Combine the different interact files via Protein Prophet
This comes from the UWPR Abacus page found here.
system("/usr/tpp_install/tpp/bin/ProteinProphet interact*.pep.xml interact-COMBINED.prot.xml")ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v5.0.0 Typhoon, Build 201612091438-exported (Linux-x86_64))
(no FPKM) (using degen pep info)
Reading in /home/srlab/Documents/ProteomicsTest/interact-20161205_sample_1.pep.xml...
...read in 0 1+, 17717 2+, 13325 3+, 2859 4+, 298 5+, 0 6+, 0 7+ spectra with min prob 0.05
Reading in /home/srlab/Documents/ProteomicsTest/interact.pep.xml...
...read in 0 1+, 17717 2+, 13325 3+, 2859 4+, 298 5+, 0 6+, 0 7+ spectra with min prob 0.05
Initializing 26114 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating protein lengths and molecular weights from database gigas-proteome.fasta
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000
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.........:.........:.........:.........:.........:.........:.........:.........:.........:........ Total: 25982
Computing degenerate peptides for 5078 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 5262 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing 4730 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating sensitivity...and error tables...
Computing MU for 5262 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%INFO: mu=8.44703e-06, db_size=23302460
Finished. Results written to: /home/srlab/Documents/ProteomicsTest/interact-COMBINED.prot.xmlStep 5: Abacus
This part is a little sketchy, as it requires a parameters file that is unique to each run (It sets up the directory structure used). I posted a sample one I used for this particular test and one can change it to fit their needs, or one can run the .jar file with the GUI (requires physical access to the machine/a VNC connection) to create your own parameters file. File here
system("java -Xmx16g -jar /home/shared/abacus/abacus.jar -p ~/Documents/ProteomicsTest/Abacus_parameters.txt")