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Interpretation of proteomics identification results
http://compomics.github.io/projects/peptide-shaker.html
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Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" #496

Open MigRibe opened 1 year ago

MigRibe commented 1 year ago

I'm trying to use the search gui and peptide shaker for peptide mass fingerprinting. Both seem pretty intuitive and complete, but I can't make progress on some algorithms in the search gui, but mostly in the peptide shaker. Attached figure. Could you help me, please? Capturar Capturar2

hbarsnes commented 1 year ago

If PeptideShaker is stuck at that particular step for long, you can cancel the process and have a look at the PeptideShaker log file. You can find it from the PeptideShaker Welcome dialog > Settings & Help > Help > Bug Report. If you share that file here we can hopefully figure out what is going on.

MigRibe commented 1 year ago

Yes, PeptideShaker is stuck at “Importing PSMs from glutenmgf.comet.pep.xml.gz”. Bug log in appendix. Many thanks

Enviado do Correiohttps://go.microsoft.com/fwlink/?LinkId=550986 para Windows

De: Harald @.> Enviado: 15 de novembro de 2022 16:42 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

Não costuma receber e-mails de @.*** Saiba por que motivo isto é importantehttps://aka.ms/LearnAboutSenderIdentification

If PeptideShaker is stuck at that particular step for long, you can cancel the process and have a look at the PeptideShaker log file. You can find it from the PeptideShaker Welcome dialog > Settings & Help > Help > Bug Report. If you share that file here we can hopefully figure out what is going on.

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1315581916, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI77JRGUAAD6SCGWDTSDWIO4OXANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

Tue Nov 15 15:07:23 GMT 2022: PeptideShaker version 2.2.17. Memory given to the Java virtual machine: 6524764160. Total amount of memory in the Java virtual machine: 128974848. Free memory: 85150896. Java version: 1.8.0_341. 1714 script command tokens (C) 2009 Jmol Development Jmol Version: 12.0.43 2011-05-03 14:21 java.vendor: Oracle Corporation java.version: 1.8.0_341 os.name: Windows 10 memory: 68.2/163.1 processors available: 8 useCommandThread: false

Tue Nov 15 15:16:37 GMT 2022: PeptideShaker version 2.2.17. Memory given to the Java virtual machine: 6524764160. Total amount of memory in the Java virtual machine: 128974848. Free memory: 82539344. Java version: 1.8.0_341. 1714 script command tokens (C) 2009 Jmol Development Jmol Version: 12.0.43 2011-05-03 14:21 java.vendor: Oracle Corporation java.version: 1.8.0_341 os.name: Windows 10 memory: 66.3/163.1 processors available: 8 useCommandThread: false

Tue Nov 15 15:17:36 GMT 2022: PeptideShaker version 2.2.17. Memory given to the Java virtual machine: 6524764160. Total amount of memory in the Java virtual machine: 128974848. Free memory: 85192296. Java version: 1.8.0_341. 1714 script command tokens (C) 2009 Jmol Development Jmol Version: 12.0.43 2011-05-03 14:21 java.vendor: Oracle Corporation java.version: 1.8.0_341 os.name: Windows 10 memory: 66.2/163.1 processors available: 8 useCommandThread: false

Tue Nov 15 15:18:28 GMT 2022: PeptideShaker version 2.2.17. Memory given to the Java virtual machine: 6524764160. Total amount of memory in the Java virtual machine: 128974848. Free memory: 83195552. Java version: 1.8.0_341. 1714 script command tokens (C) 2009 Jmol Development Jmol Version: 12.0.43 2011-05-03 14:21 java.vendor: Oracle Corporation java.version: 1.8.0_341 os.name: Windows 10 memory: 66.3/163.1 processors available: 8 useCommandThread: false https://www.ebi.ac.uk/pride/ws/archive/project/count org.springframework.web.client.HttpClientErrorException$NotFound: 404 : "{ "timestamp" : 1668525575132, "status" : 404, "error" : "Not Found", "message" : "No message available", "path" : "/pride/ws/archive/v2/project/count"}" at org.springframework.web.client.HttpClientErrorException.create(HttpClientErrorException.java:113) at org.springframework.web.client.DefaultResponseErrorHandler.handleError(DefaultResponseErrorHandler.java:168) at org.springframework.web.client.DefaultResponseErrorHandler.handleError(DefaultResponseErrorHandler.java:122) at org.springframework.web.client.ResponseErrorHandler.handleError(ResponseErrorHandler.java:63) at org.springframework.web.client.RestTemplate.handleResponse(RestTemplate.java:819) at org.springframework.web.client.RestTemplate.doExecute(RestTemplate.java:777) at org.springframework.web.client.RestTemplate.execute(RestTemplate.java:711) at org.springframework.web.client.RestTemplate.getForEntity(RestTemplate.java:361) at eu.isas.peptideshaker.gui.pride.PrideReshakeGUI$33.run(PrideReshakeGUI.java:2523)

Tue Nov 15 15:40:44 GMT 2022: PeptideShaker version 2.2.17. Memory given to the Java virtual machine: 6524764160. Total amount of memory in the Java virtual machine: 128974848. Free memory: 113195176. Java version: 1.8.0_341. java.io.IOException: Server returned HTTP response code: 500 for URL: https://rest.uniprot.org/taxonomy/sort=score&desc=&compress=no&query=Triticum aestivum&format=tab&columns=id at sun.net.www.protocol.http.HttpURLConnection.getInputStream0(Unknown Source) at sun.net.www.protocol.http.HttpURLConnection.getInputStream(Unknown Source) at sun.net.www.protocol.https.HttpsURLConnectionImpl.getInputStream(Unknown Source) at com.compomics.util.experiment.biology.taxonomy.mappings.UniprotTaxonomy.downloadMapping(UniprotTaxonomy.java:123) at com.compomics.util.experiment.biology.taxonomy.mappings.UniprotTaxonomy.getId(UniprotTaxonomy.java:177) at com.compomics.util.parameters.identification.advanced.GeneParameters.setBackgroundSpeciesFromFastaSummary(GeneParameters.java:160) at eu.isas.peptideshaker.cmd.PeptideShakerCLI.createProject(PeptideShakerCLI.java:1207) at eu.isas.peptideshaker.cmd.PeptideShakerCLI.call(PeptideShakerCLI.java:243) at eu.isas.peptideshaker.cmd.PeptideShakerCLI.main(PeptideShakerCLI.java:1467) java.io.IOException: Server returned HTTP response code: 500 for URL: https://rest.uniprot.org/taxonomy/sort=score&desc=&compress=no&query=Triticum turgidum subsp. durum&format=tab&columns=id at sun.net.www.protocol.http.HttpURLConnection.getInputStream0(Unknown Source) at sun.net.www.protocol.http.HttpURLConnection.getInputStream(Unknown Source) at sun.net.www.protocol.https.HttpsURLConnectionImpl.getInputStream(Unknown Source) at com.compomics.util.experiment.biology.taxonomy.mappings.UniprotTaxonomy.downloadMapping(UniprotTaxonomy.java:123) at com.compomics.util.experiment.biology.taxonomy.mappings.UniprotTaxonomy.getId(UniprotTaxonomy.java:177) at com.compomics.util.parameters.identification.advanced.GeneParameters.setBackgroundSpeciesFromFastaSummary(GeneParameters.java:160) at eu.isas.peptideshaker.cmd.PeptideShakerCLI.createProject(PeptideShakerCLI.java:1207) at eu.isas.peptideshaker.cmd.PeptideShakerCLI.call(PeptideShakerCLI.java:243) at eu.isas.peptideshaker.cmd.PeptideShakerCLI.main(PeptideShakerCLI.java:1467)

Tue Nov 15 16:47:27 GMT 2022: PeptideShaker version 2.2.17. Memory given to the Java virtual machine: 6524764160. Total amount of memory in the Java virtual machine: 128974848. Free memory: 83616136. Java version: 1.8.0_341. 1714 script command tokens (C) 2009 Jmol Development Jmol Version: 12.0.43 2011-05-03 14:21 java.vendor: Oracle Corporation java.version: 1.8.0_341 os.name: Windows 10 memory: 66.3/163.1 processors available: 8 useCommandThread: false

hbarsnes commented 1 year ago

I don't see any errors in the log file related to the progress halting when importing the PSMs. Would it be possible for you to share the SearchGUI output file with me so that I can try to reproduce the issue on my end?

MigRibe commented 1 year ago

Of course. Many thanks.

Enviado do Correiohttps://go.microsoft.com/fwlink/?LinkId=550986 para Windows

De: Harald @.> Enviado: 15 de novembro de 2022 21:48 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

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I don't see any errors in the log file related to the progress halting when importing the PSMs. Would it be possible for you to share the SearchGUI output file with me so that I can try to reproduce the issue on my end?

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1315904007, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI73UYGE5JYUSY7I7TUTWIQAJBANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

MigRibe commented 1 year ago

searchgui_out.zip

hbarsnes commented 1 year ago

Thanks for sharing the SearchGUI zip file! However, in order to try loading the data I will also need the spectrum and FASTA files. Any chance you could share those as well?

MigRibe commented 1 year ago

Hi, Link with raw file and fasta. Thanks https://we.tl/t-9f50pFS7Lf

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De: Harald @.> Enviado: 16 de novembro de 2022 20:20 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

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Thanks for sharing the SearchGUI zip file! However, in order to try loading the data I will also need the spectrum and FASTA files. Any chance you could share those as well?

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1317617951, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI74QZQRRB2XNAETSYRLWIU6ZLANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

hbarsnes commented 1 year ago

Thanks again! Any chance you can share the mgf file C:\Gui\MS\Glutenmgf.mgf instead of the raw file? (As I'm having some issues with the spectrum titles not matching yours if I do the conversion myself.)

MigRibe commented 1 year ago

https://we.tl/t-Jc3VXihQPr Many thanks

hbarsnes commented 1 year ago

Your data initially loaded fine on my end without any issues. However, when I downgraded my Java version to the same as the one you are using, I was finally able to reproduce the issue. I would therefore recommend that you upgrade your Java version to a more recent version and see if that solves the problem. You can find the most recent versions here: https://adoptium.net/temurin/releases. After upgrading, you can check and set the Java version used in PeptideShaker via the Welcome dialog > Settings & Help > Settings > Java Settings.

By the way, it seems like your FASTA file does not contain any decoy sequences? Without this PeptideShaker will not be able to calculate the FDR for your dataset and you cannot be sure which identifications to trust. Decoys can easily be added in SearchGUI when selecting a FASTA file. Simply click yes to the question of whether you would like to add decoys. Note that you would then have to repeat the search before later loading the data in PeptideShaker.

MigRibe commented 1 year ago

Dear Harald, Thanks for your email. I will try with other java version as soon as possible, and I will give you my feedback. Yes, I am aware, but I am trying everything to get the program to work.

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De: Harald @.> Enviado: 16 de novembro de 2022 22:45 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

Your data initially loaded fine on my end without any issues. However, when I downgraded my Java version to the same as the one you are using, I was finally able to reproduce the issue. I would therefore recommend that you upgrade your Java version to a more recent version and see if that solves the problem. You can find the most recent versions here: https://adoptium.net/temurin/releases. After upgrading, you can check and set the Java version used in PeptideShaker via the Welcome dialog > Settings & Help > Settings > Java Settings.

By the way, it seems like your FASTA file does not contain any decoy sequences? Without this PeptideShaker will not be able to calculate the FDR for your dataset and you cannot be sure which identifications to trust. Decoys can easily be added in SearchGUI when selecting a FASTA file. Simply click yes to the question of whether you would like to add decoys. Note that you would then have to repeat the search before later loading the data in PeptideShaker.

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1317774358, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI73E3AAOQPT45LWDRJ3WIVPW5ANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

MigRibe commented 1 year ago

Dear Harald, I followed your advice and I managed to get to the end of the peptideshaker analysis using the comet. Many thanks! As for Amanda, it seems to have stucked; about 4 hours waiting without leaving 33% in peptideshaker (the file is the same). The bug log is attached. PeptideShaker 2.2.17 log.txt

hbarsnes commented 1 year ago

Great to hear that you can now load the Comet data!

As for the MS Amanda issue, I don't see any explanations for this in the log file. Would you be able to share the SearchGUI zip file again?

MigRibe commented 1 year ago

Dear Harald Many thanks for your reply. https://we.tl/t-fgazVhaiAE

Also, I tried a search on another computer, but the step to open peptideshaker fails. And I can't open the file manually. I leave the log. PeptideShaker.log

hbarsnes commented 1 year ago

Aha, so it's not PeptideShaker that fails when loading the data (as for the Comet output), but MS Amanda itself that fails when searching the data? Not much we can do there I'm afraid. I would recommend having a closer look at your search parameters to see if there is anything you can consider changing there? If that does not help, you can always try contacting the MS Amanda developers directly at https://groups.google.com/g/msamanda.

Also, I tried a search on another computer, but the step to open peptideshaker fails. And I can't open the file manually. I leave the log.

I don't see anything in the error log that can explain this. What do you mean specifically with "the step to open peptideshaker fails" though?

MigRibe commented 1 year ago

Thanks for your reply! Yes, it looks like amanda gets stuck on a specific step. If I can't fix the situation maybe I'll get in touch with the amanda developers. Regarding the error on another computer, everything seems to go well (searchgui), but then there is a failure and the ongoing process (searchgui + peptideshaker) is interrupted.

Enviado do Correiohttps://go.microsoft.com/fwlink/?LinkId=550986 para Windows

De: Harald @.> Enviado: 21 de novembro de 2022 11:42 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

Aha, so it's not PeptideShaker that fails when loading the data (as for the Comet output), but MS Amanda itself that fails when searching the data? Not much we can do there I'm afraid. I would recommend having a closer look at your search parameters to see if there is anything you can consider changing there? If that does not help, you can always try contacting the MS Amanda developers directly at https://groups.google.com/g/msamanda.

Also, I tried a search on another computer, but the step to open peptideshaker fails. And I can't open the file manually. I leave the log.

I don't see anything in the error log that can explain this. What do you mean specifically with "the step to open peptideshaker fails" though?

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1321928105, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI7623FOBTLMYFDMCNLTWJNNZZANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

hbarsnes commented 1 year ago

Regarding the error on another computer, everything seems to go well (searchgui), but then there is a failure and the ongoing process (searchgui + peptideshaker) is interrupted.

You can always try running the two tools separately, i.e. first run only SearchGUI and the later try to load the data in PeptideShaker. This can perhaps provide you with a bit more information about what is happening.

MigRibe commented 1 year ago

Yes, I've tried that, but I can't open the file. Here are the files, please give me your feedback on what might be happening. https://we.tl/t-vIRghEVjRJ

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De: Harald @.> Enviado: 21 de novembro de 2022 18:51 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

Regarding the error on another computer, everything seems to go well (searchgui), but then there is a failure and the ongoing process (searchgui + peptideshaker) is interrupted.

You can always try running the two tools separately, i.e. first run only SearchGUI and the later try to load the data in PeptideShaker. This can perhaps provide you with a bit more information about what is happening.

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1322505053, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI72K6HQ27E6ACVEYIMTWJPADBANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

hbarsnes commented 1 year ago

Seems like you shared the wrong mzml file? At least PeptideShaker is asking for ModA1_GlucoseWBA.mzML and you only shared ModC1_GlioxalWBA.mzML?

MigRibe commented 1 year ago

Sorry. Please try with this https://we.tl/t-a9ZytTVJ8C

Enviado do Correiohttps://go.microsoft.com/fwlink/?LinkId=550986 para Windows

De: Harald @.> Enviado: 22 de novembro de 2022 13:26 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

Seems like you shared the wrong mzml file? At least PeptideShaker is asking for ModA1_GlucoseWBA.mzML and you only shared ModC1_GlioxalWBA.mzML?

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1323676691, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI74NQIICXMPNMCO4SADWJTCXBANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

hbarsnes commented 1 year ago

I'm now able to process your data but it ends in the following message:

Tue Nov 22 15:20:38 CET 2022        Importing sequences from UniprotSprotTD_concatenated_target_decoy.fasta.
Tue Nov 22 15:20:50 CET 2022        Importing gene mappings.
Tue Nov 22 15:22:06 CET 2022        Establishing local database connection.
Tue Nov 22 15:22:06 CET 2022        Reading identification files.
Tue Nov 22 15:22:06 CET 2022        Parsing ModA1_GlucoseWBA.comet.pep.xml.gz.
Tue Nov 22 15:22:06 CET 2022        Checking spectra for ModA1_GlucoseWBA.comet.pep.xml.gz.
Tue Nov 22 15:22:06 CET 2022        Importing PSMs from ModA1_GlucoseWBA.comet.pep.xml.gz
Tue Nov 22 15:22:09 CET 2022        666 identified spectra (78.9%) did not present a valid peptide.
Tue Nov 22 15:22:09 CET 2022        11136 of the best scoring peptides were excluded by the import filters:
Tue Nov 22 15:22:09 CET 2022            - 72.1% peptide mapping to both target and decoy.
Tue Nov 22 15:22:09 CET 2022            - 27.9% peptide length less than 8 or greater than 30.
Tue Nov 22 15:22:09 CET 2022        Warning: More than 75% of the PSMs did not pass the import filters.
 Apparently your database contains a high degree of shared peptides between the target and decoy sequences. Please verify your database.
 Please verify that your peptide selection criteria are not too restrictive.

Tue Nov 22 15:22:09 CET 2022        No identification results.

I would recommend having a look at your search settings and your FASTA file.

As for why you are not able to get to the message above, could it be that you are running out of space or memory?

MigRibe commented 1 year ago

Dear Harald, Many thanks for your help. I already got the process to come to an end in peptideshaker. I gathered the petideshaker folders that were somewhat dispersed, and updated them in the resources settings.

Some doubts:

Enviado do Correiohttps://go.microsoft.com/fwlink/?LinkId=550986 para Windows

De: Harald @.> Enviado: 22 de novembro de 2022 14:27 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

I'm now able to process your data but it ends in the following message:

Tue Nov 22 15:20:38 CET 2022 Importing sequences from UniprotSprotTD_concatenated_target_decoy.fasta.

Tue Nov 22 15:20:50 CET 2022 Importing gene mappings.

Tue Nov 22 15:22:06 CET 2022 Establishing local database connection.

Tue Nov 22 15:22:06 CET 2022 Reading identification files.

Tue Nov 22 15:22:06 CET 2022 Parsing ModA1_GlucoseWBA.comet.pep.xml.gz.

Tue Nov 22 15:22:06 CET 2022 Checking spectra for ModA1_GlucoseWBA.comet.pep.xml.gz.

Tue Nov 22 15:22:06 CET 2022 Importing PSMs from ModA1_GlucoseWBA.comet.pep.xml.gz

Tue Nov 22 15:22:09 CET 2022 666 identified spectra (78.9%) did not present a valid peptide.

Tue Nov 22 15:22:09 CET 2022 11136 of the best scoring peptides were excluded by the import filters:

Tue Nov 22 15:22:09 CET 2022 - 72.1% peptide mapping to both target and decoy.

Tue Nov 22 15:22:09 CET 2022 - 27.9% peptide length less than 8 or greater than 30.

Tue Nov 22 15:22:09 CET 2022 Warning: More than 75% of the PSMs did not pass the import filters.

Apparently your database contains a high degree of shared peptides between the target and decoy sequences. Please verify your database.

Please verify that your peptide selection criteria are not too restrictive.

Tue Nov 22 15:22:09 CET 2022 No identification results.

I would recommend having a look at your search settings and your FASTA file.

As for why you are not able to get to the message above, could it be that you are running out of space or memory?

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1323762565, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI73FC2KIIAHIPWHGGRLWJTJ6PANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

hbarsnes commented 1 year ago

the X! tandem says the spectrum doesn't meet the necessary criteria, but I've changed several settings and it still doesn't work. I did the test with the same spectrum using the X! tandem on Petunia and did the analysis. The X! tandem is the one from the latest release?

The version included in SearchGUI is X! TANDEM Vengeance (2015.12.15.2). The specific search engine settings can be found in the resources\temp\search_engines folder during the running of the search (but are removed when the search is completed). Perhaps a direct comparison of the parameters will indicate why there are differences in the results.

Also be sure to check out the advanced X! Tandem settings by clicking the cog wheel next to X! Tandem in the main SearchGUI dialog.

when looking for modifications in peptides, if there is more than one modification in an amino acid, peptideshaker fails in the visualization and in the coverage bar; with just one modification there is no problem.

Generally, more than one modification is not supported on the same reside unless it is at the terminals. Can you share an example where you have two modifications on the same residue (that is not at the termini)? As these should have been filtered out when loading the data in PeptideShaker. And can you also share the error log for when the front end fails?

I also noticed some differences when using comet on peptideshaker and petunia. Peptideshaker does not detect fragments from charge greater than 3. Is there any reason why the results are different using the same search criteria?

You would again have to compare the specific search settings. But the default value for the maximum fragment charge for Comet is 3. You may however change this in the advanced Comet settings by clicking the cog wheel next to Comet in the main SearchGUI dialog.

I ams also interested in identifying cross-linked peptides. Is there any routine you recommend me in searchgui+peptideshaker? Is there a possibility for petideshaker to work with Kojak?

The identification of crosslinked peptides is not support by the search engines included in SearchGUI. Or at least not implemented in SearchGUI at the moment. I have not looked into this in detail, but as far as I remember only MetaMorpheus has support for this (https://github.com/smith-chem-wisc/MetaMorpheus/wiki/Crosslink-Search-Task), however not from inside SearchGUI (nor PeptideShaker).

finally, I would like to ask you if a workstation substantially reduces the search time, or rather what requirements you would recommend. We have some searches taking more than 2 hours (i7 10th series, 16 gb ram, ssd).

This depends on what you mean by search. If you are talking about the processing in PeptideShaker, the main limiting factor is the amount of memory. Note that you will have to set this manually in the PeptideShaker Welcome dialog > Settings & Help > Settings > Java Settings > Memory. Increasing the number of CPUs can also speed up the processing as then you can utilize more parallel processing.

When it comes to the searches in SearchGUI we have much less control over how the individual search engines utilize the resources provided, but generally the same reasoning applies, i.e. the more memory and CPUs the better.

MigRibe commented 1 year ago

Hi, thanks for your reply,

Generally, more than one modification is not supported on the same reside unless it is at the terminals. Can you share an example where you have two modifications on the same residue (that is not at the termini)? As these should have been filtered out when loading the data in PeptideShaker. And can you also share the error log for when the front end fails? The potential modifications found by the program can actually happen as we are using experimental models to check chemical derivatizations. So, what happens is that the same peptide (in terms of identity) can have different modifications in the same amino acid from the attribution of different scans. When this happens, the location on the protein is the same (bottom bar) and it gives an error when you click there. At the moment I can't have the files. If it's important I'll try to upload it.

Other questions:

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De: Harald @.> Enviado: 30 de novembro de 2022 15:18 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

the X! tandem says the spectrum doesn't meet the necessary criteria, but I've changed several settings and it still doesn't work. I did the test with the same spectrum using the X! tandem on Petunia and did the analysis. The X! tandem is the one from the latest release?

The version included in SearchGUI is X! TANDEM Vengeance (2015.12.15.2). The specific search engine settings can be found in the resources\temp\search_engines folder during the running of the search (but are removed when the search is completed). Perhaps a direct comparison of the parameters will indicate why there are differences in the results.

Also be sure to check out the advanced X! Tandem settings by clicking the cog wheel next to X! Tandem in the main SearchGUI dialog.

when looking for modifications in peptides, if there is more than one modification in an amino acid, peptideshaker fails in the visualization and in the coverage bar; with just one modification there is no problem.

Generally, more than one modification is not supported on the same reside unless it is at the terminals. Can you share an example where you have two modifications on the same residue (that is not at the termini)? As these should have been filtered out when loading the data in PeptideShaker. And can you also share the error log for when the front end fails?

I also noticed some differences when using comet on peptideshaker and petunia. Peptideshaker does not detect fragments from charge greater than 3. Is there any reason why the results are different using the same search criteria?

You would again have to compare the specific search settings. But the default value for the maximum fragment charge for Comet is 3. You may however change this in the advanced Comet settings by clicking the cog wheel next to Comet in the main SearchGUI dialog.

I ams also interested in identifying cross-linked peptides. Is there any routine you recommend me in searchgui+peptideshaker? Is there a possibility for petideshaker to work with Kojak?

The identification of crosslinked peptides is not support by the search engines included in SearchGUI. Or at least not implemented in SearchGUI at the moment. I have not looked into this in detail, but as far as I remember only MetaMorpheus has support for this (https://github.com/smith-chem-wisc/MetaMorpheus/wiki/Crosslink-Search-Task), however not from inside SearchGUI (nor PeptideShaker).

finally, I would like to ask you if a workstation substantially reduces the search time, or rather what requirements you would recommend. We have some searches taking more than 2 hours (i7 10th series, 16 gb ram, ssd).

This depends on what you mean by search. If you are talking about the processing in PeptideShaker, the main limiting factor is the amount of memory. Note that you will have to set this manually in the PeptideShaker Welcome dialog > Settings & Help > Settings > Java Settings > Memory. Increasing the number of CPUs can also speed up the processing as then you can utilize more parallel processing.

When it comes to the searches in SearchGUI we have much less control over how the individual search engines utilize the resources provided, but generally the same reasoning applies, i.e. the more memory and CPUs the better.

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1332333835, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI74AXWPYB62I542CZMTWK5V5FANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

hbarsnes commented 1 year ago

The potential modifications found by the program can actually happen as we are using experimental models to check chemical derivatizations. So, what happens is that the same peptide (in terms of identity) can have different modifications in the same amino acid from the attribution of different scans. When this happens, the location on the protein is the same (bottom bar) and it gives an error when you click there. At the moment I can't have the files. If it's important I'll try to upload it.

Aha, so what you are talking about are different PSMs for the same peptide having different PTMs on the same residue? That can of course happen. I thought you were talking about more than one PTM on the same residue for the same PSM. I guess we just never came across any such cases ourselves. Would be great if you could point me to one such example. Maybe you can also share the error log for when this happens?

How peptideshaker performs merging of the results of different search algorithms?

It basically compares all of the matches for the same spectrum across the search engines and picks the one with the best score.

Is it possible to use a random database (scrambled decoys) instead reverse decoys for FDR calculation?

I think you can create your decoys any way you want as long as they are properly annotated in the FASTA headers. I'm not sure if we have ever done any testing with scrambled decos though. In general there is however no real difference between reversed and scrambled decoys with regards to the results. Hence I would not bother with this unless you have very good reasons to do so.

How peptideshaker uses data from for example two replicates? (i.e. adding two samples at once) does it merge all the spectra?

If results from more than one spectrum file are loaded at the same time the results are indeed merged into one common result. If you want to look at the two replicates separately you will therefore have to load them individually. You can however check out the Fractions tab as it would give you some ideas of which peptides that were detected in each spectrum file, but this is generally intended for loading multiple fractions from the same sample and not for comparing different replicates or samples.

MigRibe commented 11 months ago

Dear Harald Barsnes, After some time that I didn't use the program I did it again, but the peptide shaker fails to open. I send the report and I was able to locate the file .psdb. Can you please help me? Raw file at https://we.tl/t-lTvc6otxXP

I would also like to ask you if it is possible to do a research on adducts, but for a polymer-type adduct. That is, let's imagine that I have a monomer linked to an amino acid, is it possible to search for different species of [x]n polymers?

Best Miguel

@.*** https://molecules4life-2023.events.chemistry.pt/

De: Harald @.> Enviado: 12 de dezembro de 2022 10:57 Para: @.> Cc: José Miguel Silva Ferreira @.>; @.> Assunto: Re: [compomics/peptide-shaker] Peptide-shaker doesn't progress from "Importing PSMs from mgf.comet.pep.xml.gz" (Issue #496)

The potential modifications found by the program can actually happen as we are using experimental models to check chemical derivatizations. So, what happens is that the same peptide (in terms of identity) can have different modifications in the same amino acid from the attribution of different scans. When this happens, the location on the protein is the same (bottom bar) and it gives an error when you click there. At the moment I can't have the files. If it's important I'll try to upload it.

Aha, so what you are talking about are different PSMs for the same peptide having different PTMs on the same residue? That can of course happen. I thought you were talking about more than one PTM on the same residue for the same PSM. I guess we just never came across any such cases ourselves. Would be great if you could point me to one such example. Maybe you can also share the error log for when this happens?

How peptideshaker performs merging of the results of different search algorithms?

It basically compares all of the matches for the same spectrum across the search engines and picks the one with the best score.

Is it possible to use a random database (scrambled decoys) instead reverse decoys for FDR calculation?

I think you can create your decoys any way you want as long as they are properly annotated in the FASTA headers. I'm not sure if we have ever done any testing with scrambled decos though. In general there is however no real difference between reversed and scrambled decoys with regards to the results. Hence I would not bother with this unless you have very good reasons to do so.

How peptideshaker uses data from for example two replicates? (i.e. adding two samples at once) does it merge all the spectra?

If results from more than one spectrum file are loaded at the same time the results are indeed merged into one common result. If you want to look at the two replicates separately you will therefore have to load them individually. You can however check out the Fractions tab as it would give you some ideas of which peptides that were detected in each spectrum file, but this is generally intended for loading multiple fractions from the same sample and not for comparing different replicates or samples.

— Reply to this email directly, view it on GitHubhttps://github.com/compomics/peptide-shaker/issues/496#issuecomment-1346270447, or unsubscribehttps://github.com/notifications/unsubscribe-auth/A4GQI76JO3X5YRMWTFPCTZ3WM4AKFANCNFSM6AAAAAASBD2I6U. You are receiving this because you authored the thread.Message ID: @.***>

------------------------------------------------------------------

SearchGUI 4.2.17 Report File

#

Originally saved by: Miguel Ribeiro @ Miguel-PC

on: 02 ago. 2023, 12:26

as: Report

------------------------------------------------------------------

Wed Aug 02 12:19:24 WEST 2023 Formatting uniprotkb_proteome_UP000019116_2023_07_27_concatenated_target_decoy.fasta for OMSSA.

Building a new DB, current time: 08/02/2023 12:19:24 New DB name: uniprotkb_proteome_UP000019116_2023_07_27_concatenated_target_decoy.fasta New DB title: uniprotkb_proteome_UP000019116_2023_07_27_concatenated_target_decoy.fasta Sequence type: Protein Keep Linkouts: T Keep MBits: T Maximum file size: 1073741824B Adding sequences from FASTA; added 261346 sequences in 7.67394 seconds.

Wed Aug 02 12:19:31 WEST 2023 Database Formating finished for uniprotkb_proteome_UP000019116_2023_07_27_concatenated_target_decoy.fasta (7.9 seconds).

Wed Aug 02 12:19:31 WEST 2023 uniprotkb_proteome_UP000019116_2023_07_27_concatenated_target_decoy.fasta formatted for OMSSA.

Wed Aug 02 12:19:31 WEST 2023 Converting raw files. Wed Aug 02 12:19:31 WEST 2023 Processing F1.raw with ThermoRawFileParser.

2023-08-02 12:19:32 INFO Started parsing C:\Users\Miguel Ribeiro\Desktop\LCMS Spots\F\F1.raw 2023-08-02 12:19:33 INFO Processing 20140 MS scans

2023-08-02 12:19:39 INFO Finished parsing C:\Users\Miguel Ribeiro\Desktop\LCMS Spots\F\F1.raw 2023-08-02 12:19:39 INFO Processing completed 0 errors, 0 warnings

Wed Aug 02 12:19:39 WEST 2023 ThermoRawFileParser finished for F1.raw (7.6 seconds).

Wed Aug 02 12:19:39 WEST 2023 Importing spectrum files. Wed Aug 02 12:19:39 WEST 2023 Importing spectrum file F1.mzml Wed Aug 02 12:19:41 WEST 2023 Importing spectrum files completed (2.1 seconds).

Wed Aug 02 12:19:41 WEST 2023 Converting spectrum file F1.mzml to peak list.

Wed Aug 02 12:19:42 WEST 2023 Processing F1.mgf with OMSSA.

Info: Using 8 search threads Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough 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omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: omssa: not enough peaks in spectra Info: Sequence=0 Percent=0% Info: Sequence=10000 Percent=3.82635% Info: Sequence=20000 Percent=7.65269% Info: Sequence=30000 Percent=11.479% Info: Sequence=40000 Percent=15.3054% Info: Sequence=50000 Percent=19.1317% Info: Sequence=60000 Percent=22.9581% Info: Sequence=70000 Percent=26.7844% Info: Sequence=80000 Percent=30.6108% Info: Sequence=90000 Percent=34.4371% Info: Sequence=100000 Percent=38.2635% Info: Sequence=110000 Percent=42.0898% Info: Sequence=120000 Percent=45.9161% Info: Sequence=130000 Percent=49.7425% Info: Sequence=140000 Percent=53.5688% Info: Sequence=150000 Percent=57.3952% Info: Sequence=160000 Percent=61.2215% Info: Sequence=170000 Percent=65.0479% Info: Sequence=180000 Percent=68.8742% Info: Sequence=190000 Percent=72.7006% Info: Sequence=200000 Percent=76.5269% Info: Sequence=210000 Percent=80.3532% Info: Sequence=220000 Percent=84.1796% Info: Sequence=230000 Percent=88.0059% Info: Sequence=240000 Percent=91.8323% Info: Sequence=250000 Percent=95.6586% Info: Sequence=260000 Percent=99.485%

Wed Aug 02 12:22:55 WEST 2023 OMSSA finished for F1.mgf (3 minutes 13.0 seconds).

Wed Aug 02 12:22:55 WEST 2023 Zipping output files.

Wed Aug 02 12:22:55 WEST 2023 Processing identification files with PeptideShaker.

Wed Aug 02 12:24:45 WEST 2023 PeptideShaker processing canceled. Please see the PeptideShaker log file: C:\Users\Miguel Ribeiro\Desktop\PeptideShaker-2.2.25\PeptideShaker-2.2.25\resources\PeptideShaker.log

Wed Aug 02 12:24:45 WEST 2023 Searching Canceled! C:\Users\Miguel Ribeiro\Desktop\PeptideShaker-2.2.25\PeptideShaker-2.2.25\resources\matches\F120230802-122307.psdb could not be deleted! Wed Aug 02 12:25:05 WEST 2023 The search or processing did not finish properly!

hbarsnes commented 11 months ago

After some time that I didn't use the program I did it again, but the peptide shaker fails to open. I send the report and I was able to locate the file .psdb. Can you please help me?

Can you please also share the PeptideShaker log file: C:\Users\Miguel Ribeiro\Desktop\PeptideShaker-2.2.25\PeptideShaker-2.2.25\resources\PeptideShaker.log?

I would also like to ask you if it is possible to do a research on adducts, but for a polymer-type adduct. That is, let's imagine that I have a monomer linked to an amino acid, is it possible to search for different species of [x]n polymers?

I guess you could create your own polymer adduct modifications and search for those. However you would have to create one per x and include each in your search. So I guess it depends on how high x would be. If x is, for example, 2 or 3 you should be ok, but if x is much higher you probably need a custom search engine. I do not have any experience with such searches though. Perhaps it would be worth asking some of the search engine developers directly? See for example: https://groups.google.com/g/comet-ms.