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compomics / searchgui

Highly adaptable common interface for proteomics search and de novo engines
http://compomics.github.io/projects/searchgui.html
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TMT results in SearchCLI 4.0.0 #260

Closed chrishuges closed 4 years ago

chrishuges commented 4 years ago

Hi,

I am having an odd issue in version 4.0.0 of SearchCLI/PeptideShaker 2.0.0. Basically, if I search a file with TMT labeling, I get zero, or very close to zero hits in the output files. If I search a normal, unlabeled analysis file, I have no problems and the result looks good.

I have tried this with files from TMT 11-plex and 16-plex labeled samples. In the case of the 11-plex samples, these give good results in the previous versions of SearchCLI. I have tried putting the peptide N-terminal label in the fixed or variable spots, and it doesn't make a difference (TMT on K is always variable).

There are no errors in either the SearchCLI or PeptideShakerCLI log files. I have tried this with XTandem and Comet and had the same result with both.

Example XTandem output:

Wed Nov 04 17:13:37 PST 2020 Importing spectrum files.
Wed Nov 04 17:13:37 PST 2020 Importing spectrum files completed (28.0 milliseconds).

Processing: ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf (1/1)
Wed Nov 04 17:13:37 PST 2020 Converting spectrum file ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf to peak list.
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xtandem command:
/projects/ptx_analysis/chughes/software/SearchGUI-4.0.0/resources/XTandem/linux/linux_64bit/tandem /projects/ptx_analysis/chughes/software/SearchGUI-4.0.0/resources/XTandem/linux/linux_64bit/input_searchGUI.xml

Wed Nov 04 17:13:43 PST 2020 Processing ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf with X!Tandem.

X! TANDEM Vengeance (2015.12.15.2)

Loading spectra (mgf)....... loaded.
Spectra matching criteria = 12115
Starting threads ................................ started.
Computing models:
        Spectrum-to-sequence matching process in p      sequences modelled = 41 ks
Model refinement:
        partial cleavage  done.
        unanticipated cleavage  done.
        finishing refinement ... done.
Merging results:
        from 234567891011121314151617181920212223242526272829303132

Creating report:
        initial calculations  ..... done.
        sorting  ..... done.
        finding repeats ..... done.
        evaluating results ..... done.
        calculating expectations ..... done.
        writing results ..... done.

Valid models = 0

Wed Nov 04 17:16:35 PST 2020 X!Tandem finished for ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf (2 minutes 53.0 seconds).

Wed Nov 04 17:16:36 PST 2020 Zipping output files.

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Wed Nov 04 17:16:36 PST 2020 Search Completed (2 minutes 59.0 seconds).

Search Completed.

No peptide shaker reports output exists, so it will be created.
Wed Nov 04 17:16:37 PST 2020 Unzipping ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.rawsearchgui_out.zip.
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Wed Nov 04 17:16:38 PST 2020 Import process for 05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8

WARNING: An illegal reflective access operation has occurred
WARNING: Illegal reflective access by com.esotericsoftware.kryo.unsafe.UnsafeUtil (file:/projects/ptx_analysis/chughes/software/PeptideShaker-2.0.0/lib/kryo-5.0.0-RC9.jar) to method sun.nio.ch.DirectBuffer.cleaner()
WARNING: Please consider reporting this to the maintainers of com.esotericsoftware.kryo.unsafe.UnsafeUtil
WARNING: Use --illegal-access=warn to enable warnings of further illegal reflective access operations
WARNING: All illegal access operations will be denied in a future release
Wed Nov 04 17:16:39 PST 2020 Importing sequences from uniprotHumanCrapTargetDecoyNov2020.fasta.
Wed Nov 04 17:16:39 PST 2020 Importing gene mappings.
Wed Nov 04 17:16:40 PST 2020 Establishing local database connection.
Wed Nov 04 17:16:40 PST 2020 Reading identification files.
Wed Nov 04 17:16:40 PST 2020 Parsing ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.t.xml.gz.
10%
Wed Nov 04 17:16:40 PST 2020 No PSM found in ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.t.xml.gz.
Wed Nov 04 17:16:40 PST 2020 No identification results.
Wed Nov 04 17:16:40 PST 2020 PeptideShaker Processing Canceled.
<CompomicsError>PeptideShaker processing canceled. Please see the PeptideShaker log file: /projects/ptx_analysis/chughes/software/PeptideShaker-2.0.0/resources/PeptideShaker.log</CompomicsError>

Example Comet output:

Wed Nov 04 17:00:54 PST 2020 Importing spectrum files.
Wed Nov 04 17:00:54 PST 2020 Importing spectrum files completed (17.0 milliseconds).

Processing: ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf (1/1)

comet command:
/projects/ptx_analysis/chughes/software/SearchGUI-4.0.0/resources/Comet/linux/comet.exe /projects/ptx_results/2019/Lumos-VCP/07-Jul/Samples/CH/dataProcessing_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8/ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf

Wed Nov 04 17:00:54 PST 2020 Processing ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf with Comet.

 Comet version "2019.01 rev. 5"
 Search start:  11/04/2020, 05:00:54 PM
 - Input file: /projects/ptx_results/2019/Lumos-VCP/07-Jul/Samples/CH/dataProcessing_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8/ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf
   - Load spectra: 12135
     - Search progress:                                                                                 100%
     - Post analysis:  done
 Search end:    11/04/2020, 05:02:10 PM, 1m:16s

Wed Nov 04 17:02:12 PST 2020 Comet finished for ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.mgf (1 minute 18.0 seconds).

Wed Nov 04 17:02:12 PST 2020 Zipping output files.

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Wed Nov 04 17:02:14 PST 2020 Search Completed (1 minute 20.0 seconds).

Search Completed.

No peptide shaker reports output exists, so it will be created.
Wed Nov 04 17:02:15 PST 2020 Unzipping ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.rawsearchgui_out.zip.
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Wed Nov 04 17:02:16 PST 2020 Import process for 05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8

WARNING: An illegal reflective access operation has occurred
WARNING: Illegal reflective access by com.esotericsoftware.kryo.unsafe.UnsafeUtil (file:/projects/ptx_analysis/chughes/software/PeptideShaker-2.0.0/lib/kryo-5.0.0-RC9.jar) to method sun.nio.ch.DirectBuffer.cleaner()
WARNING: Please consider reporting this to the maintainers of com.esotericsoftware.kryo.unsafe.UnsafeUtil
WARNING: Use --illegal-access=warn to enable warnings of further illegal reflective access operations
WARNING: All illegal access operations will be denied in a future release
Wed Nov 04 17:02:16 PST 2020 Importing sequences from uniprotHumanCrapTargetDecoyNov2020.fasta.
Wed Nov 04 17:02:17 PST 2020 Importing gene mappings.
Wed Nov 04 17:02:17 PST 2020 Establishing local database connection.
Wed Nov 04 17:02:17 PST 2020 Reading identification files.
Wed Nov 04 17:02:17 PST 2020 Parsing ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.comet.pep.xml.gz.
Wed Nov 04 17:02:20 PST 2020 Checking spectra for ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.comet.pep.xml.gz.
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Wed Nov 04 17:02:20 PST 2020 Importing PSMs from ch_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.raw.comet.pep.xml.gz
 50% 60% 70% 80% 90% 100% 110% 120% 130% 140%
Wed Nov 04 17:02:22 PST 2020 206 identified spectra (1.8%) did not present a valid peptide.
Wed Nov 04 17:02:22 PST 2020 17309 of the best scoring peptides were excluded by the import filters:
Wed Nov 04 17:02:22 PST 2020     - 80.5% peptide presenting high mass or isotopic deviation.
Wed Nov 04 17:02:22 PST 2020     - 19.4% unrecognized modifications.
Wed Nov 04 17:02:22 PST 2020 File import completed. 11767 first hits imported (114470 total) from 12134 spectra.
Wed Nov 04 17:02:22 PST 2020 [11557 first hits passed the initial filtering]
Wed Nov 04 17:02:22 PST 2020 Computing assumptions probabilities.
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Wed Nov 04 17:02:22 PST 2020 Saving assumptions probabilities, selecting best match, scoring modification localization.
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Wed Nov 04 17:02:27 PST 2020 Computing PSM probabilities.
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Wed Nov 04 17:02:27 PST 2020 Resolving peptide inference issues.
Wed Nov 04 17:02:27 PST 2020 Peptide Inference. Please Wait...
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Wed Nov 04 17:02:29 PST 2020 Saving probabilities, building peptides and proteins.
Wed Nov 04 17:02:29 PST 2020 Saving Probabilities. Building Peptides and Proteins. Please Wait...
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Wed Nov 04 17:02:30 PST 2020 Generating peptide map.
Wed Nov 04 17:02:30 PST 2020 Filling Peptide Maps. Please Wait...
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Wed Nov 04 17:02:31 PST 2020 Computing peptide probabilities.
Wed Nov 04 17:02:31 PST 2020 Saving peptide probabilities.
Wed Nov 04 17:02:31 PST 2020 Attaching Peptide Probabilities. Please Wait...
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Wed Nov 04 17:02:32 PST 2020 Simplifying protein groups.
Wed Nov 04 17:02:32 PST 2020 Symplifying Protein Groups. Please Wait...
Wed Nov 04 17:02:32 PST 2020 65 unlikely protein mappings found:
Wed Nov 04 17:02:32 PST 2020     - 47 protein groups supported by predicted or uncertain proteins.
Wed Nov 04 17:02:32 PST 2020     - 18 protein groups explained by simpler groups.
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Wed Nov 04 17:02:32 PST 2020 Mapping shared peptides.
Wed Nov 04 17:02:32 PST 2020 Inferring PI Status and Sorting Proteins. Please Wait...
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Wed Nov 04 17:02:33 PST 2020 Generating protein map.
Wed Nov 04 17:02:33 PST 2020 Filling Protein Map. Please Wait...
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Wed Nov 04 17:02:34 PST 2020 Selecting leading proteins, inferring peptide and protein inference status.
Wed Nov 04 17:02:34 PST 2020 Inferring PI Status and Sorting Proteins. Please Wait...
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Wed Nov 04 17:02:34 PST 2020 Computing protein probabilities.
Wed Nov 04 17:02:35 PST 2020 Saving protein probabilities.
Wed Nov 04 17:02:35 PST 2020 Attaching Protein Probabilities. Please Wait...
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Wed Nov 04 17:02:36 PST 2020 Validating identifications at 1% FDR, quality control of matches.
Wed Nov 04 17:02:36 PST 2020 Finding FDR Thresholds. Please Wait...
Wed Nov 04 17:02:36 PST 2020 Match Validation and Quality Control. Please Wait...
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Wed Nov 04 17:02:37 PST 2020 Scoring PTMs in peptides.
Wed Nov 04 17:02:37 PST 2020 Scoring Peptide Modification Localization. Please Wait...
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Wed Nov 04 17:02:38 PST 2020 Estimating spectrum counting scaling values.
Wed Nov 04 17:02:39 PST 2020 Scoring PTMs in proteins, gathering summary metrics.
Wed Nov 04 17:02:39 PST 2020 Scoring Protein Modification Localization. Please Wait...
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Wed Nov 04 17:02:40 PST 2020 Identification processing completed (23.6 seconds).

Wed Nov 04 17:02:40 PST 2020 Project successfully created.
Wed Nov 04 17:02:40 PST 2020 Saving results.
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Wed Nov 04 17:02:43 PST 2020 Results saved to /projects/ptx_results/2019/Lumos-VCP/07-Jul/Samples/CH/dataProcessing_05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8/05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8.out.psdb.

Wed Nov 04 17:02:44 PST 2020 PeptideShaker process completed.
Wed Nov 04 17:02:44 PST 2020 Processing Completed.
WARNING: An illegal reflective access operation has occurred
WARNING: Illegal reflective access by com.esotericsoftware.kryo.unsafe.UnsafeUtil (file:/projects/ptx_analysis/chughes/software/PeptideShaker-2.0.0/lib/kryo-5.0.0-RC9.jar) to method sun.nio.ch.DirectBuffer.cleaner()
WARNING: Please consider reporting this to the maintainers of com.esotericsoftware.kryo.unsafe.UnsafeUtil
WARNING: Use --illegal-access=warn to enable warnings of further illegal reflective access operations
WARNING: All illegal access operations will be denied in a future release
Wed Nov 04 17:02:46 PST 2020 Exporting. Please Wait...
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Wed Nov 04 17:02:52 PST 2020 Report export completed.

Finished all exporting reports for 05July2019_CHLA9-CHLA10-HEK293_TMT9_HpH_frac8 data set.

Finished processing a total of 1 files!

The modifications file looks OK to me:

"marshallableParameterType": "identification_parameters",
  "version": "5.0.1",
  "name": "databaseSearchParameters",
  "description": "Variable: Oxidation of M, TMT 11-plex of K, TMT 11-plex of peptide N-term.\nPrecursor Tolerance: 20.0 ppm.\nFragment Tolerance: 0.5 ppm.\n",
  "defaultDescription": true,
  "searchParameters": {
    "version": "5.0.1",
    "precursorAccuracyType": "PPM",
    "fragmentAccuracyType": "PPM",
    "precursorTolerance": 20.0,
    "fragmentIonMZTolerance": 0.5,
    "modificationParameters": {
      "fixedModifications": [],
      "variableModifications": [
        "Oxidation of M",
        "TMT 11-plex of K",
        "TMT 11-plex of peptide N-term"
      ],
      "refinementVariableModifications": [],
      "refinementFixedModifications": [],
      "colors": {},
      "backUp": {
        "TMT 11-plex of K": {
          "modificationType": "modaa",
          "name": "TMT 11-plex of K",
          "shortName": "TMT",

Any idea what might be happening here?

Thanks, Chris

hbarsnes commented 4 years ago

Hi Chris,

I just tried with a 10-plex TMT dataset and did not see any such issues. I don't have any 11- or 16-plex datasets available though, so would it be possible for you to share these so that I can try to locate the problem? You can send them directly to me at harald.barsnes@gmail.com.

Best regards, Harald

hbarsnes commented 4 years ago

Hi again,

One thing I note is that you are using a fragment ion accuracy of only 0.5 ppm. Is this in purpose? Perhaps you can try with something like 0.02 Da?

Best regards, Harald

chrishuges commented 4 years ago

Hi Harald,

Ah, that was silly of me. You are correct, the fragment mass error was too small because it was set to ppm. Was this default as 'Da' in previous releases? I don't remember having to specify it as such before. In any case, it is working fine now.

Thanks for looking at that. Chris

hbarsnes commented 4 years ago

Hi Chris,

Yes, I think the default was Dalton in the previous releases. Not sure why it was changed. But great to hear that you were able to get it to work.

Best regards, Harald