SRM Analysis Part 7
Another Skyline saga
To figure out what was going on with my technical replicates, Emma said she’d take a look at my Skyline document. Here’s what she found:
I found some discrepancies between your settings and settings I had for one of my SRM files (one of which was that you told Skyline you had DIA data…). I’ve updated the settings and I am re-importing your data. In peptide settings, the 13C modifications weren’t checked (PRTC modifications). In transition settings, mine looked like the attached, which were different from yours. I think the most significant would be the Full-scan settings.
I’m not sure why my Skyline settings were off! Emma and I made the Skyline document together, but we may not have actually adjusted the settings. Here are my old, incorrect Skyline settings:
Figures 1-11. Old Skyline settings
And here are the actual settings:
Figures 12-22. Revised Skyline settings
Once again, I fixed all transitions and ensured all peaks were picked correctly using my old Skyline document. Emma was unable to find any chromatograms in samples 14 and 15.
Figures 23-26. Examples of transitions that needed to be fixed.
Figure 27. NMDS for technical replication using nonnormalized data.
Figure 28. NMDS for technical replication using normalized data.
Now I just need to figure out how to analyze and present my SRM data for PCSGA…………