Hi Theresa,
We spoke by WebEx, and I hope it became clear that Skyline is definitely designed with the intent of supporting what you are trying to achieve, but that you had gotten somewhat off the track we have designed and hoped you would use.
In the end, we came up with an approach that should get you most of the way to what you were hoping to achieve, with some extra manual work still required for your non-tryptic peptides.
The high-level workflow can be summarized as:
1. Create a broad target list covering your proteins of interest.
2. Use Edit > Refine > Accept Peptides to narrow the list to only the peptides you are interested in and all of their modified forms.
In our first attempt on the phone call we did this without any spectral library filtering and ended up with around 18,000 modified forms of your the 143 peptides out of your 160 desired that were fully-tryptic and not too long for your settings.
Then, because you mentioned the Global Proteome Machine (GPM) as the source of your information on what to monitor we started downloading GPM libraries directly from the FTP site (
ftp://ftp.thegpm.org/projects/xhunter/libs/eukaryotes/lib/human_chromosomes/), which we, unfortunately, had to do 1 chromosome at a time. (I got through 3, 5, 11, 15, 16 and 20.) With those plus the NIST Human library I got 428 modified peptide forms with 549 precursors. After applying Edit > Refine > Accept Peptides with your 160 peptide list, I was left with 102 modified peptide forms with 131 precursors.
See attached PowerPoint slides for an overview of the steps we took.
Thanks for the quick WebEx this morning.
--Brendan