Spectral library with modifications

[cutleraging]

Hello Vadim and Team,

I am using a spectral library for modified histone peptides that I built in skyline. I have some questions regarding using this library...

1. What is the best way to convert the skyline spectral library (.blib) to a format that will work with DIA-NN? So far I was able to make a .tsv file by following these recommendations: https://skyline.ms/announcements/home/support/thread.view?rowId=65491. I have also attached it for your reference.
2. When I use this spectra library I encounter this warning: "WARNING: below are the unknown modifications found in the library. While unlikely to have a signficant effect, these might negatively affect identification performance in certain scenarious, it is strongly recommended to declare them using any of --mod, --fixed-mod or --var-mod: +112.1, +126.1, +14, +142.1, +170.1, +226.1, +28, +56, +70, +84.1, +86, +98, +98.1." Since I am looking at histone peptides, there are both fixed and variable modifications. Although this information is all in the spectral library. Is it necessary to specify the modifications as stated in the warning? It appears in my output that the modified peptides were still able to be identified (attached).
3. When creating spectral libraries, is it always best to use DDA? Or should I use the method that I plan to use for quantification (e.g. slicePASEF).

231123_SC_histones_100cell_spectral_library_DIA-NN_edited.txt

skyline spectral library with 100 cells test.txt

Best, Ronnie