Skyline CMD help

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Skyline CMD help smanda  2020-08-13
 

Hi Brendon/Kaipo,

I am trying to build a spectral library all using command line as we are trying to automate some of the steps at our lab. To this, I am unable to figure out, how can I choose peptides standards for RT calibration (after I import the library). My current steps are:

  1. SkylineCmd.exe --in=Empty.sky --out=New_library.sky --import-search-file=searchresults.mzid --import-fasta=20190717_Uniprot_concat_decoys.fasta
    This step creates a nr library and adds the peptides as targets.
    I do not specify RT peptides here.
    I then import a report format

  2. SkylineCmd.exe" --report-add=skyline.skyr

  3. SkylineCmd.exe --in=New_library.sky --report-name=OpenSWATH --report-file=test_report.tsv --report-format=tsv

I see that the exported reported as several NAs in the iRT column. We have around 40 endogenous peptides that are added as iRT standards in all experiments, can you please let me know, how to I add them at RT peptides and export the report with iRT values.

Regards,
Srikanth

 
 
Kaipo responded:  2020-08-20

Hi Srikanth,

This functionality should've been available from the command line, but we overlooked it. It will be available soon (likely next month).

Thanks,
Kaipo

 
smanda responded:  2020-09-29

Hi Kaipo,

Any updates on this feature? Is it now already available in command line?

Regards,
Srikanth

 
Kaipo responded:  2020-09-29

Hi Srikanth,
I've implemented this feature and should have it merged before the next Skyline-daily release.

Thanks,
Kaipo

 
smanda responded:  2020-10-13

Hi Kaipo,

Thanks to you I see an update which says Skyline-daily 20.2.1.286 has the iRT implementation. Can you please let me know with an example command how to use the feature?

Regards,
Srikanth

 
Kaipo responded:  2020-10-13

Hi Srikanth,
You can see the documentation for the command line through Help > Documentation > Command Line. The new options are under the "Importing peptide searches" section.

As an example:
SkylineCmd.exe --in=exampledocument.sky --import-search-file=results.mzid --import-search-irts=Biognosys-11 (iRT-C18)
The created document library (in this case exampledocument.blib) will function as an iRT database and the document will have the RT predictor added.

Note that if using CiRT peptides as iRT standards, the --import-search-num-cirts=<integer> option must be included with how many peptides to use as standards.

Thanks,
Kaipo

 
smanda responded:  2020-10-13

Hi Kaipo,

Thanks for the quick response. What I am still not clear is about specifying own iRTs. I guess, thats the CiRTs.
With the --import-search-irts <Name>, do I now specify the protein name which has all the ciRT peptides? I have around 40 endogenous peptides.

Regards,
Srikanth

 
Kaipo responded:  2020-10-14

Hi Srikanth,
You can add a new iRT standard in the "Edit iRT Calculator" dialog (Settings > Peptide Settings > Prediction > Edit iRT Calculator) by clicking the Calibrate button. When you add the new standard, you can name it as well. This is the name you would use with the --import-search-irts option.

Thanks,
Kaipo