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: https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_dia

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?