Hi Skyline!

I'm trying to build a spectral library using DDA files from injections ran on some real world samples with my heavy peptides spiked in. I followed the "Import Peptide Searhc resultts" workflow with the following notes

1. i did my search on MSFragger an uploaded both the interact-pep.xml and interact-.mod.pep.xml files. The MS fragger tutorial only mentioned the former but I added both jsut in case.
2. I have two types of heavy peptides: those that come from my whole protein standard (uniformly N15 labeled, no other PTMs) and my AQUA peptides (a single heavy Valine w/ 6.0135 Da mass shift). These injections were either the N15 whole protein digest alone, or with AQUA peptides spiked in.

Becuase I know what standards are in each injection, I know what the ground truth is and while I do get some false hits in my MSFragger searches, it is properly identifying my heavy peptides. However, when I go through the wizard following your tutorial, my Skyline document has lots of fake peptides like the one in the first attached image. However, for the samples that Skyline says it is "present", the peak look perfectly real but I know that I don't have any standards with a phospho at that position.

I attached another example where Skyline didn't automatically add this peptide but I went in to add it. Note that it gives me "real peaks" for the light peptide but as you can see from the attached chromatogram, the heavy elutes at 20 minutes and the light @ 14. A 6 minute discrepancy. From my understanding, if the light peptide is really there it should also elute @ 20 min.

Would you reccomend making two different libraries for injections with my whole protein standard (uniform N15 label) and my AQUA peptides (one heavy valine)?

Ultimately, my goal is to be able to filter through my data and pick what ions I'll ultimately use to do all my work whether I do this anaylsis with DIA or PRM. Any help is appreicated! Please let me know what extra information I can provide to fix the issue.