Spectral library building with open source/free MS/MS identification software Erik  2021-04-21
 

Hi MacCoss-members,

I've been trying different free/open-source MS2 identifcation software to identify my MS2 spectra from my thermo altis triple quadrupole (only MS2 no MS1) and use their output for a Skyline spectral library.

So far I've tried Peptideshaker/SearchGUI with X-Tandem, MSAmanda, comet, omssa and msgf+ without any success. Somehow the mzid/pepxml/mzIdentML are not able to be read by the Skyline spectral library builder.

Tried maxquant but it does not work because I think it needs MS1 spectra.
Tried PD 2.4 demo (with MSAmanda and Sequest) and that does work but it will expire soon.

Any suggestions on how to import the formats from any of the free search engines properly?

Greetings,
Erik de Graaf

PS. It would be great if you could integrate a search engine like MSAmanda to build a spectral library for targeted assay development!

 
 
Kaipo responded:  2021-04-21

Hi Erik,
Those sources should work with Skyline. What error are you getting from Skyline when building the spectral library? Would you mind sharing an example of files that are not working for you (either attached to this request or uploaded to https://skyline.ms/files.url)?

Thanks,
Kaipo

 
Erik responded:  2021-04-22
Hi Kaipo,

Here is the messages I got when trying to import the pep.xml from comet, the mzml from msgf+ and the mzid from peptideshaker.

First the comet pep.xml file with the mzML together in one directory and gives this error below.



---------------------------
Skyline
---------------------------
ERROR: Invalid cvParam accession "1003145".
ERROR: PwizReader could not parse .\20210408_TSQALTIS_UM2_Graaf168_SA_SN10824024_PTA-013_MSMS.mzML

Command-line: C:\Users\ErikdeGraafPepscope\AppData\Local\Apps\2.0\2328QLZT.01Q\9LTA2RM4.Z08\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0 -i comet -S "C:\Users\ErikdeGraafPepscope\AppData\Local\Temp\tmp7B64.tmp" "C:\Temp\SearchGUI\Comet-MSGFplus-Unimod\comet.redundant.blib"
Working directory: C:\Temp\SearchGUI\Comet-MSGFplus-Unimod
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: Invalid cvParam accession "1003145".
ERROR: PwizReader could not parse .\20210408_TSQALTIS_UM2_Graaf168_SA_SN10824024_PTA-013_MSMS.mzML

Command-line: C:\Users\ErikdeGraafPepscope\AppData\Local\Apps\2.0\2328QLZT.01Q\9LTA2RM4.Z08\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0 -i comet -S "C:\Users\ErikdeGraafPepscope\AppData\Local\Temp\tmp7B64.tmp" "C:\Temp\SearchGUI\Comet-MSGFplus-Unimod\comet.redundant.blib"
Working directory: C:\Temp\SearchGUI\Comet-MSGFplus-Unimod
   bij pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:regel 62
   bij pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:regel 201
   bij pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:regel 156
---------------------------

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Then below is an example of the msgf+ output created by search gui.
I have the msgf+ *.mzid file with the *.mzML together in one directory and gives this error below.

---------------------------
Skyline
---------------------------
ERROR: Invalid cvParam accession "1003145".
ERROR: PwizReader could not parse .\20210408_TSQALTIS_UM2_Graaf168_SA_SN10824024_PTA-013_MSMS.mzML

Command-line: C:\Users\ErikdeGraafPepscope\AppData\Local\Apps\2.0\2328QLZT.01Q\9LTA2RM4.Z08\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0 -i msgfplus -S "C:\Users\ErikdeGraafPepscope\AppData\Local\Temp\tmpCB18.tmp" "C:\Temp\SearchGUI\Comet-MSGFplus-Unimod\msgfplus.redundant.blib"
Working directory: C:\Temp\SearchGUI\Comet-MSGFplus-Unimod
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: Invalid cvParam accession "1003145".
ERROR: PwizReader could not parse .\20210408_TSQALTIS_UM2_Graaf168_SA_SN10824024_PTA-013_MSMS.mzML

Command-line: C:\Users\ErikdeGraafPepscope\AppData\Local\Apps\2.0\2328QLZT.01Q\9LTA2RM4.Z08\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0 -i msgfplus -S "C:\Users\ErikdeGraafPepscope\AppData\Local\Temp\tmpCB18.tmp" "C:\Temp\SearchGUI\Comet-MSGFplus-Unimod\msgfplus.redundant.blib"
Working directory: C:\Temp\SearchGUI\Comet-MSGFplus-Unimod
   bij pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:regel 62
   bij pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:regel 201
   bij pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:regel 156
---------------------------

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



Then there is the peptideshaker mzidentml output I tried to import with this error message:

---------------------------
Skyline
---------------------------
ERROR: Invalid cvParam accession "1003145".
ERROR: PwizReader could not parse .\20210408_TSQALTIS_UM2_Graaf168_SA_SN10824024_PTA-013_MSMS.mzML

Command-line: C:\Users\ErikdeGraafPepscope\AppData\Local\Apps\2.0\2328QLZT.01Q\9LTA2RM4.Z08\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0 -i comet_msgfplus_mzidentml -S "C:\Users\ErikdeGraafPepscope\AppData\Local\Temp\tmpABFA.tmp" "C:\Temp\SearchGUI\Comet-MSGFplus-Unimod\comet_msgfplus_mzidentml.redundant.blib"
Working directory: C:\Temp\SearchGUI\Comet-MSGFplus-Unimod
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: Invalid cvParam accession "1003145".
ERROR: PwizReader could not parse .\20210408_TSQALTIS_UM2_Graaf168_SA_SN10824024_PTA-013_MSMS.mzML

Command-line: C:\Users\ErikdeGraafPepscope\AppData\Local\Apps\2.0\2328QLZT.01Q\9LTA2RM4.Z08\skyl..tion_e4141a2a22107248_0014.0002_2f1cb11a037aa924\BlibBuild -s -A -H -o -c 0 -i comet_msgfplus_mzidentml -S "C:\Users\ErikdeGraafPepscope\AppData\Local\Temp\tmpABFA.tmp" "C:\Temp\SearchGUI\Comet-MSGFplus-Unimod\comet_msgfplus_mzidentml.redundant.blib"
Working directory: C:\Temp\SearchGUI\Comet-MSGFplus-Unimod
   bij pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:regel 62
   bij pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:regel 201
   bij pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:regel 156
---------------------------

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

Could it be due to type of raw file that only contains targeted MS/MS scans on the Altis?
I actually prefer to use MSAmanda because it gives better phosphosite localisation as output. I can also generate some files for you for this as well.

Greetings,
Erik
 
Kaipo responded:  2021-04-26
Hi Erik,

Can you try removing line 29:
<cvParam cvRef="MS" accession="MS:1003145" name="ThermoRawFileParser" />
from the mzML file and then building the library?

It looks like we need to update our CV list but until then this might be a good workaround.

Thanks,
Kaipo
 
matt.chambers42 responded:  2021-04-28
Kaipo's suggestion should work. Although if you have a lot of these files it might be better to either try searching the RAWs directly or convert to mzML with msconvert instead. The next Skyline-daily should have the updated CV which will work with ThermoRawFileParser (although it appears they actually reverted their change to use the newer cvParam for exactly this reason).
 
Erik responded:  2021-04-29
Hi Kaipo and Matt,

Thanks for the support.
Both solutions work for the comet pepxml files.

Thanks,
Erik

PS. Are MSAmanda mzid files generated from the MSAmanda standalone not supported?