Mirrorplotting measured MS/MS spectra against library spectra?

Mirrorplotting measured MS/MS spectra against library spectra? lackmann  2021-08-11

I did a targeted analysis in Skyline using a library build using Prosit as a data source (Library --> Build --> Prosit). All worked out great, but as a final quality control and as a figure in a presentation, I want to compare MS/MS spectra from my runs against the library spectra, following the old View --> MS/MS spectra option. However, when I use View --> Library Match I can only generate plots between Prosit and my library (conveniently, I get a perfect dotp score as my Prosit-build library spectrum is compared with, well, Prosit).
Nevertheless, such a mirror plot with a measured MS/MS spectrum against the library would be great, although I would also settle for the old style without the mirror option, but it seems to no longer exist or I am just unable to find it. Search function sadly does not help as the old View --> MS/MS spectrum document is still online under "Tips", so maybe someone could point me in the right direction...?

Thank you in advance,

Brendan MacLean responded:  2021-08-11

View > Library Match is just the old View > MS/MS Spectrum renamed.

Where are your measured spectra? Are they in a different library than the one you generated from Prosit? If so, then I think you have 2 options:

  1. Uncheck the right-click > Prosit menu item, but leave "Mirror" checked. This should then give you a toolbar option allowing you to choose which libraries to mirror against each other, and you can mirror your Prosit library against your empirically measured library.
  2. Either remove the Prosit library (you can Undo this operation) or make it second in the Peptide Settings > Library order. This should get Skyline choosing your empirical spectra above the Prosit library spectra, and then mirroring with dynamically queried Prosit spectra in the Library Match view should give you what you are asking for.

The Targeted Method Editing tutorial covers working with multiple libraries (though, not with Prosit).

If your empirical MS/MS spectra are not in a separate library, then I will need to understand better what you are trying to achieve.

Thanks for posting to the Skyline support board.


lackmann responded:  2021-08-11

Thanks for the quick reply Brendan!

In the current project, I only have a Prosit-based library build directly in Skyline and the results of a SureQuant targeted assay are imported with via import --> results. For most of my targets, results look great. However, as additional control (and to understand what happened with the not-so-great targets), I also checked the original MS/MS files and now would like to see how they differ from the Prosit prediction (and maybe make a nice figure out of it...).
So in Skyline I am trying to take a look at the empirical MS/MS spectra chosen by Skyline for my product traces, mirror plot with my library spectra would be nice but not necessary, I can also build something from scratch there. Reasoning for going by Skyline would be so no doubts are that the spectra presented are really the same as shown in the Skyline chromatogram traces.

Thanks again for your great support and the great software that is Skyline,