Command line SRL generation

Command line SRL generation smanda  2020-01-20

Hi Brendon,

I have search engines results as mzid file which I import into Skyline and create a spectral library. I then map the peptides to proteins and export the library as a OpenSWATH report. Is it possible to do all these steps using the command line? I have several of these and interested to automate the process of library generation using Skyline.

  1. Import mzid and clean (probably bibliospec does this)
  2. Calcualte iRTs using standards
  3. Map peptides to proteins
  4. Import into project and export as a report.

Any help appreciated.


Brendan MacLean responded:  2020-01-21

Yes, that should all be possible. Have you looked at the command-line help which you can get to from Help > Documentation > Command Line, or by running SkylineCmd or SkylineRunner with no arguments or --help?

smanda responded:  2020-01-28

Hi Brendan,

Thanks for your reply. I had a look at the SkylineCMD and was wondering if you could help me with some questions.

  1. the --in parameter seems to be compulsory, does it mean I create and empty project inside skyline and use that as input? Does it mean I cannot create a skyline document without GUI?
  2. importing peptide searches and creating library, these has to be two separate commands or will work in single command, not clear?
  3. Same is case with exporting. Each header in the documentation are separate set of commands for individual execution or can be run simultaneously? How can I replicate my settings used in GUI, if I have saved them?


Brendan MacLean responded:  2020-01-29

Hi Srikanth,

  1. Yes, some initial file is expected. The command-line does not offer enough control over the settings to make it reasonable for you to start with the default settings and create an entire document without starting from some kind of template. However, the current functionality should be sufficient for you to start with a template document which contains only initial settings (Peptide, Transition, Document Settings) and no targets.
  2. Should be possible in one command. You can also use --batch to perform many operations in a single command.
  3. Command arguments can be combined in various ways. You can definitely use --in --out --import --export all in one command. A good place to look at how the command line can be applied is webinars 14 and 15, which come with batch scripts that make use of SkylineCmd/SkylineRunner. Download from the webinar pages ( and

Hope this helps. You should also just start to play around with small files, like the tutorial files to understand better what is possible.