SILAC channels not showing up during DIA analysis

SILAC channels not showing up during DIA analysis dghandour  2023-08-24 19:27

Hello! I'm relatively new to Skyline and this is my first time doing a DIA analysis, so I would appreciate some guidance. I'm running on Windows, Skyline daily.

I'm currently working on a proteomics DIA analysis. My process began with generating a few DDA runs on our Bruker timsTOF and running them through FragPipe for building a spectral library, and then generating a set of DIA runs (also on our Bruker timsTOF). The samples have a mix of light, medium, and heavy SILAC labeled proteins (R and K, R[6] and K[6], R[10] and K[8]).

After that, I followed the Skyline guide for spectral library generation and DIA analysis almost exactly (skipping the isolation window creation part).
Here's what I was following:

Although SILAC isn't explicitly discussed in the manual, I account for the SILAC channels during my analysis, in the Modifications section. When the data comes out, it shows two channels, a "light" and a "heavy", where the heavy channel probably accounts for both the medium and the heavy SILAC labels. (See "pic 1")

When I go to peptide settings and designate the correct labels to "medium" and "heavy" SILAC (see "pic 2"), then I go back to the chromatograms, nothing shows up for the medium or heavy SILAC channels. It says that there's no chromatogram available, for every peptide. (See "pic 3")

When I do all these SILAC settings for a DDA analysis, it usually works great. Is there something I'm doing wrong on Skyline, or do you think it's a problem with the original pep.xml files I used for making the spectral library?

Nick Shulman responded:  2023-08-24 20:40
You might be able to get chromatograms to show up if you go to:
Edit > Manage Results
select all the replicates and push the "Reimport" button.

If the missing chromatograms do not show up when you do that, then you should send us your Skyline document and one or more of your raw files and we will figure out what is going on.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

You can also zip up one or more of your Bruker .d folders.
Files which are less than 50MB can be attached to this support request.
You can upload larger files here:

If you have done a reimport and you are still seeing "Chromatogram information unavailable" then it usually means that none of the spectra in the mass spec data file matched the precursor in your document, which might have something to do with the settings at "Settings > Transition Settings > Full Scan" or it might be that no matching spectra were acquired in your experiment.

We will probably be able to figure out what is going on when we see your files.
-- Nick
dghandour responded:  2023-08-25 16:55
Hey Nick,

I think I may have figured it out! In Peptide Settings, it was using the wrong library, and wasn't using the one I originally made at the beginning of the process. After I switched to that library and re-imported, I started seeing medium and heavy. Thank you for your help!