Hi,
I cannot figure out why my Skyline document is missing results for some fragments, and Skyline's behaviour appears somewhat inconsistent to me.

The setup is a bit unusual, but I hope the following makes sense.

• I am interested in peptides with two K residues and these can be either 13C or 12C. I have therefore applied a variable isotope label and used Refine -> Permute Isotope Modifications -> Complete. This results in 4 precursors per peptide (both lysines labelled, no lysine labelled, the first lysine labelled, the second lysine labelled).
• For other reasons, my cells were grown on 13C glucose, all other amino acids therefore carry fixed structural modifications to make them 13C
• Samples were measured on a Sciex QTOF 6600 with a targeted PRM method generated by Skyline.

The problem is as follows: The two precursors with exactly one labelled and one unlabelled lysine have exactly the same precursor mass, but they do have unique fragments (fragments which contain exactly one lysine). I would therefore like to use these for quantification to distinguish the two label states. When I import my .wiff data, Skyline shows no result for some fragments. And it seems to somehow recognise which fragments are shared and unique between the two precursors. Sometimes it shows no data for fragments which are shared (which is weird but OK for me), but in other cases it excludes all unqie fragments (which is bad).

Do you have any insight why it is not extracting results for some of these fragments? I realise this is a somewhat off-label use of Skyline but hopefully there is a workaround. I have attached a Skyline file with two peptides. The interesting thing is happening with the heavy2 precursor where some fragments are not quantified. For the first peptide the unqiue fragments are not quantified, for the second fragment, the shared fragments are not quantified.

Thanks a lot for your help,
Stephan