Hi Donna,
- Is there a smarter/more efficient way to remove these peptides?
I would usually use Edit > Refine > Advanced - Min transitions per precursor for this. You just specify a minimum like 3, if you have no MS1 precursors, or 6 if you have 3 MS1 precursor transitions in every peptide. You can also set this up from the start by setting Transition Settings - Library - Minimum product ions. We would typically recommend at least 4 and my understanding is that the Aebersold lab often sets both Pick: [ ] product ions and [ ] minimum product ions to 6. Using either method to keep from getting precursors with less than 3 fragment ions will keep you document from having precursors where the library dot product cannot be calculated.
- Do you have any idea what could cause this? Maybe more generally, could you give me an idea why a peptide would not meet this criteria? And last but not least: how can I deal with this?
I don't know why these peptides would end up missing in your iRT library, but in a fractionated library, I suppose, if the iRT peptides could not be detected sufficiently in one of the fractions and that fraction did not otherwise contain enough overlapping peptides with any of the other fractions, that could force Skyline to abandon the effort to give its peptides iRT values and you might end up with detections in your library without corresponding iRT values. Just a guess, though. I would be happy to look further, if I could get the files.
To recover I would open the iRT Calculator editor on your current iRT library (Peptide Settings - Prediction - click the button with the calculator icon > Edit Current) Select all of the peptides in your iRT calculator (maybe even adding the iRT peptides? using a text editor) Then use Edit > Refine > Accept Peptides and past the list into this form. This should reduce your document to only peptides with iRT values.
You could also use the Document Grid, which now allows deleting selected elements. So you would build a report for peptides with a retention time prediction column, filter for everything where the prediction is #N/A, select all, and click the delete button.
- Is the file size that I have somewhat as expected? The output .skyd-file is around 380GB, which sounds like a lot to me?
That does sound pretty big. It would likely get smaller and the processing faster once you only have peptides with iRT values. When you use peptides without iRT values, Skyline is forced to extract full gradient chromatograms. But, 208,000 peptides with, say, 10 chromatograms each (3 precursors, 7 fragments) could end up around 2,000,000 chromatograms with 60 files. That could get pretty big. Though, 380 GB still sounds larger than I would expect. Fix your other problems first and we will see where you end up. Are you otherwise, using +/- 5 minutes around RT predictions? Or something else?
Hope this helps as a start.
--Brendan