Hi, much like everyone else here, I should probably mention how incredibly useful Skyline has been for me. I've been using it for the last 2.5 years, starting as a learning tool for MS and now using it for my own projects as a graduate student. That being said, I would probably still consider myself a beginner with this software. Right now I'm using Skyline to help me quantify changes in histone ptms, and it's not an understatement to say that I couldn't do it without this software. However, I'm running into a weird issue related to MS2 spectra not being recognized in Skyline.
I'm running Skyline Daily 19.1.1.248 on Windows 10.
I'll start by detailing my workflow:
- I run my samples on a bruker QTOF using a DDA workflow. I've manually added certain masses to a mass preference list so that some ions would fragment at all points during their elution. This is to facilitate quantification of co-eluting isobaric ions. (I'll admit, it's possible that this step isn't working as intended, but it wouldn't explain all the behavior I'm seeing).
- I convert my files to centroided .mzml files and use Metamorpheus to calibrate and run a search for modified peptides. I import the resulting .mzid files into skyline as a spectral library with a cutoff of 0.99. My peptide settings allow a large number of PTMs (10), but restrict peptides to those found in the library.
- I import a fasta of selected histones (just H3 for now), and remove repeated peptides.
- I import my .mzml files and use the peptide identification times on each spectra to select peaks.
All of this seems to work well. However, when I look at MS2 spectra, I notice that I have relatively few MS2 peaks, even when peptide ids are shown on the spectra. I've attached an image demonstrating this. I can click on the IDs and see my fragment ions changing, but Skyline doesn't seem to have any MS2 at those timepoints. On the image, you can see the highest scoring ion as a straight line from practically zero abundance to its max, That same ion in the ID spectra increases, but not in a straight line manner.
Noting another poster had a similar issue which was resolved by changing the MS/MS filtering to a DDA strategy, I attempted that without success.
Any help would be appreciated. I've uploaded a minimal Skyline project that replicates this as a .zip to Uploads Page (File Name: M.Mann Bug.zip). I imagine you'll need the raw files too, so I've uploaded those as well (File Name: M.Mann Raw Files.zip).
Thanks regardless! Hopefully this can be resolved easily.
Sincerely,
Morgan Mann
University of Wisconsin-Madison
Brasier Lab