Hey,
alright, I suppose I understood something very wrong then and also worded my first post badly too.
I want to use DDA runs (n=29) to build a spectral library for SWATH use in Skyline.
I did that by selecting the settings mentioned above in "peptide settings" and "transition settings". Subsequently, in "peptide settings" -> "library" -> "build library" -> "iRT standard peptides" set to the biognosys 11 as mentioned above. Input files are 29 .group files. Afterwards, I uploaded the SwissProt.fasta, filtered a bit more with "refine".
I just downloaded the Skyline-daily and redid the whole thing with MS/MS filtering for DDA. I didn't know Skyline offered that, thank you for pointing it out.
"So, you imported your DDA data into Skyline after building the library? Usually, we would recommend importing DDA data with MS1 filtering only and using ion type "p" instead of "y, b". It sounds like you are instead treating your DDA data like it was DIA or PRM data.
As you could probably tell, it works well if I use precursor filtering only. I figured I could look into DDA data with that as well but... really, in retrospect I didn't question what I was trying to do too much, I apologize for having bothered you due to my mindlessness there.
Most importantly though; thank you so much ! I'll play around some more.