in silico trypsin digestion from a FASTA file nicolas drouin  2021-05-27
 

Dear Skyline team,

I encounter a problem with the in silico digestion of my FASTA file with trypsin (KR/P).
(https://www.uniprot.org/uniprot/P0DTC2)
For unknown reasons, the first generated peptide starts at residue 77 where the first K residue is. But there should be many more peptides with the R cleavage site before this peptide.

Another problem I see is the count of the residue. Indeed, this first peptide K.RFDNPVLPFNDGVYFASTEK. Indeed, according to the fast file, the residue R is located in position 78 but skyline indicated in position 77.

I'm running the last version of skyline-daily.

Please find enclosed the skyline file I'm using.

Many thanks in advance.
Best regards,
Nicolas

 
 
Nick Shulman responded:  2021-05-27
There is a setting "Exclude N-terminal AAs" at:
Settings > Peptide Settings > Filter

Your document has that set to 45, so Skyline excludes peptides whose starting position in the protein is less than 45.

If you change that setting to a smaller number you will see the missing peptides.

The off-by-one problem with the amino acid positions was pointed out to us a few years ago. Skyline starts counting amino acid positions at "0" instead of "1", because that was what made the most sense to us as programmers. By the time that scientists pointed out that we were alone in that thinking, we felt that it was too late to change it without potentially causing problems for users who might be relying on the old behavior.
-- Nick
 
nicolas drouin responded:  2021-06-03
thank you very much !!!!!!!!!!!!!
i didn't know about this function !